Classifications

• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
  • A01N1/10—Preservation of living parts
  • A01N1/12—Chemical aspects of preservation
  • A01N1/122—Preservation or perfusion media

Definitions

• Biological materials such as proteins and nucleic acids, may in certain instances, be freeze-dried (lyophilized) to enhance their stability in the absence of refrigeration. Freeze-drying comprises freezing of an aqueous mixture containing a biological material and removal of water via sublimation. Biological materials can suffer denaturation (e.g., partial denaturation) as a result of freeze-drying, e.g., as a result of the freezing step and/or the sublimation step.
• denaturation e.g., partial denaturation
• the present disclosure provides for preservation of biological materials, such as proteins, nucleic acids and biological samples, in substantially water-free fluid media.
• biological materials such as proteins, nucleic acids and biological samples
• the water free fluid media has boiling point higher than than of water.
• the biological materials are soluble and stable in the substantially water- free fluid media at ambient temperature or higher for extended periods of time, and thus do not need to be refrigerated or frozen during shipping or storage.
• the biological materials retain their structural integrity, function and activity after preservation in the substantially water- free fluid media at ambient temperature or higher for extended periods of time. Because the biological materials are preserved in a fluid medium, they may not need to be re-dissolved for use in fluid-phase reactions or assays, including nucleic acid amplification reactions based on polymerase chain reaction (PCR) and analytical and diagnostic assays, such as immunoassays.
• PCR polymerase chain reaction
• compositions comprising a biological material in a substantially water-free fluid medium, wherein the fluid medium comprises a non-ionic organic solvent (e.g., an alcohol solvent) or an ionic organic solvent comprising an organic salt and an organic hydrogen bond donor.
• a non-ionic organic solvent e.g., an alcohol solvent
• an ionic organic solvent comprising an organic salt and an organic hydrogen bond donor.
• the compositions can further comprise a metal salt, and/or one or more substances selected from the group consisting of reducing agents, antioxidants, free radical scavengers, oxygen radical scavengers, hydroxyl radical scavengers, singlet oxygen quenchers, hydroperoxide -removing agents, protease inhibitors, nuclease inhibitors, ribonuclease (RNase) inhibitors, deoxyribonuclease (DNase) inhibitors, metal chelators, preservatives, anti-microbials, buffers (or buffering agents), detergents, and chaotropes.
• reducing agents antioxidants, free radical scavengers, oxygen radical scavengers, hydroxyl radical scavengers, singlet oxygen quenchers, hydroperoxide -removing agents, protease inhibitors, nuclease inhibitors, ribonuclease (RNase) inhibitors, deoxyribonuclease (DNase) inhibitor
• the disclosure further provides methods of preserving a biological material, comprising mixing an aqueous mixture comprising a biological material with a non-ionic organic solvent (e.g., an alcohol solvent) or an ionic organic solvent comprising an organic salt and an organic hydrogen bond donor to produce an aqueous organic mixture, and removing water from the aqueous organic mixture, e.g., by evaporation, to produce a substantially water-free fluid medium comprising the biological material and the non-ionic or ionic organic solvent.
• a non-ionic organic solvent e.g., an alcohol solvent
• an ionic organic solvent comprising an organic salt and an organic hydrogen bond donor
• the fluid medium can further comprise a metal salt and/or one or more substances as described herein.
• compositions that comprise biological materials in substantially water- free fluid media.
• Figure 1 shows electrophoresis results of reverse transcription PCR (RT-PCR) for analysis of human 18S ribosomal RNA (rRNA) after preservation of RT-PCR reagents in glycerol, with addition of no sucrose or varying amounts of sucrose, at ambient temperature for varying periods of time.
• RT-PCR reverse transcription PCR
• Figures 2 and 3 show electropherograms of multiplex PCR for analysis of all 13 short tandem repeat (STR) loci utilized in the CODIS forensic database, as well as Penta D, Penta E and amelogenin, after preservation of PCR reagents in glycerol, with addition of no sucrose or varying amounts of sucrose, at ambient temperature for varying periods of time.
• STR short tandem repeat
• Figure 4 shows mixtures containing human serum solids and varying amounts of glycerol and sucrose after removal of water under reduced pressure.
• Figure 6a-c shows the effect of dry storage of IgA, IgGl and IgM at 25°C in several high boiling point alcohol solvents for (a) 5 hrs room temperature, (b) 3 hrs room temperature and 2 hrs at 37 °C, and (c) 3 hrs room temperature and 2 hrs at 56 °C.
• Figure 7 shows quantitative recovery of serum immunoglobulin activity in all three of IgA, IgGl, IgM relative to the matched untreated serum sample (green arrow).
• Figure 8 shows preservation of PCR reagents in glycerol plus sucrose or sorbitol for STR-based human identification.
• Headings are included herein for reference and to aid in locating certain sections.
• exemplary means "serving as an example, instance, or illustration”. Any embodiment characterized herein as “exemplary” is not necessarily to be construed as preferred or advantageous over other embodiments.
• the term “ambient temperature” or “room temperature” refers to a temperature range from about 18 °C to about 27 °C, or from about 20 °C to about 25 °C, or from about 22 °C to about 25 °C. In other embodiments, the term “ambient temperature” or “room temperature” refers to a temperature of about 18 °C, 19 °C, 20 °C, 21 °C, 22 °C, 23 °C, 24 °C, 25 °C, 26 °C or 27 °C. In certain embodiments, the term “ambient temperature” or “room temperature” refers to a temperature of about 22 °C, 23 °C, 24 °C or 25 °C.
• halide refers to fluoride, chloride, bromide and iodide.
• biological reaction and “biochemical reaction” are used interchangeably herein unless expressly indicated otherwise.
• a biological material can be transferred from an aqueous medium to a substantially water-free fluid medium without passing through an intermediate solid state (e.g., without freezing of the aqueous medium or an aqueous organic medium), by mixing of an aqueous mixture comprising the biological material with a non-ionic organic solvent (e.g., an alcohol solvent) or an ionic organic solvent and removal of water (e.g., by evaporation) from the resulting aqueous organic mixture.
• a non-ionic organic solvent e.g., an alcohol solvent
• ionic organic solvent e.g., an alcohol solvent
• the fluid medium can comprise a metal salt, and/or one or more substances selected from the group consisting of reducing agents, antioxidants, free radical scavengers, oxygen radical scavengers, hydroxyl radical scavengers, singlet oxygen quenchers, hydroperoxide -removing agents, protease inhibitors, nuclease inhibitors, ribonuclease (RNase) inhibitors, deoxyribonuclease (DNase) inhibitors, metal chelators, preservatives, anti-microbials, buffers (or buffering agents), detergents, and chaotropes.
• reducing agents antioxidants, free radical scavengers, oxygen radical scavengers, hydroxyl radical scavengers, singlet oxygen quenchers, hydroperoxide -removing agents, protease inhibitors, nuclease inhibitors, ribonuclease (RNase) inhibitors, deoxyribonuclease (DNase) inhibitors
• the biological materials are soluble and stable in the substantially water-free fluid media at ambient temperature or higher for extended periods of time, and thus do not need to be refrigerated or frozen during shipping or storage.
• the biological materials retain their structural integrity, function and activity after preservation in the substantially water- free fluid media at ambient temperature or higher for extended periods of time.
• the biological materials retain their structural integrity, function and activity even though alcohol solvents (including polyol solvents) and ionic organic solvents (including deep eutectic solvents) comprising an organic salt and an organic hydrogen bond donor can denature biological materials such as proteins (including enzymes) and nucleic acids (including double-stranded DNA).
• the biological materials retain their structural integrity, function and activity even though removal of water from an aqueous organic mixture comprising a biological material (e.g., a protein, such as an enzyme) and a salt (e.g., an inorganic salt, such as sodium chloride) can result in, e.g., at least a 5-fold or 10-fold greater concentration of the salt in the substantially water- free fluid medium, which high salt concentration may be expected to be deleterious to the structure, function and/or activity of the biological material.
• a biological material e.g., a protein, such as an enzyme
• a salt e.g., an inorganic salt, such as sodium chloride
• preservation of biological materials in substantially water-free fluid media can facilitate handling of the biological materials.
• biological materials of the present disclosure are preserved in a fluid medium, they may not need to be re- dissolved for use in fluid-phase reactions or assays, including nucleic acid amplification reactions based on PCR and analytical and diagnostic assays, such as immunoassays.
• compositions comprising a biological material in an anhydrous, non-ionic organic solvent
• compositions comprising a biological material in a substantially water-free, non-ionic organic solvent (e.g., an alcohol solvent).
• a substantially water-free, non-ionic organic solvent e.g., an alcohol solvent.
• such a composition comprises:
• polypeptide comprises an enzyme that mediates a nucleic acid reaction, a polypeptide that regulates an enzyme, an antibody, a polypeptide ligand of an antibody, a polypeptide aptamer, a protein or enzyme useful for detection, a toxin, a hormone, a cytokine, a polypeptide therapeutic or a vaccine, or a derivative thereof or any combination thereof;
• the polynucleotide comprises a polynucleotide used in a nucleic acid reaction, a catalytic polynucleotide, or a polynucleotide that binds specifically to a target ligand, or a derivative thereof or any combination thereof; and wherein the biological sample comprises a biological fluid, a biological suspension, a fluid aspirate, blood, plasma, serum, lymph, cerebrospinal fluid, gastric fluid, bile, perspiration, ocular fluid, tears, oral fluid, sputum, saliva, a buccal sample, a tonsil sample, a nasal sample, mucus, a nasopharyngeal sample, semen, urine, a vaginal sample, a cervical sample, a rectal sample, a fecal sample, a wound or purulent sample, hair, a tissue, a tissue homogenate, cells, a cellular lysate, a tissue or cell biopsy, skin cells, tumor or cancer cells
• the at least one alcohol solvent in the composition comprises no more than about 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% or 0.5% water by mass relative to the combined mass of water and the at least one alcohol solvent, e.g., after storage of the composition in a closed container (e.g., a capped tube, vial or well) at a temperature from ambient temperature to about 40 °C for at least about 1 day, 3 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, 1 year, 1.5 years or 2 years.
• a closed container e.g., a capped tube, vial or well
• the at least one alcohol solvent in the composition comprises no more than about 10%, 5% or 1%> water by mass relative to the combined mass of water and the at least one alcohol solvent, e.g., after storage of the composition in a closed container (e.g., a capped tube, vial or well) at a temperature from ambient temperature to about 40 °C for at least about 1 day, 3 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, 1 year, 1.5 years or 2 years.
• a closed container e.g., a capped tube, vial or well
• the at least one alcohol solvent in the composition comprises no more than about 10%>, 5% or 1% water by mass relative to the combined mass of water and the at least one alcohol solvent after storage of the composition in a closed container (e.g., a capped tube, vial or well) at ambient temperature for at least about 3 months or 6 months.
• a closed container e.g., a capped tube, vial or well
• the at least one alcohol solvent is substantially soluble in water - e.g., at least about 50%, 60%, 70%, 80%, 90%, 95% or 99% of the at least one alcohol solvent by mass or volume is soluble in water. In certain embodiments, at least about 90%, 95% or 99% of the at least one alcohol solvent by mass or volume is soluble in water. In an embodiment, the at least one alcohol solvent is miscible with water. Solubility of the at least one alcohol solvent in water promotes transfer of a biological material from an aqueous medium to the at least one alcohol solvent.
• the at least one alcohol solvent has a boiling point substantially greater than that of water - e.g., a boiling point at least or greater than about 110 °C, 125 °C, 150 °C, 175 °C, 200 °C or 250 °C at a pressure of about 1 atmosphere (atm). In certain embodiments, the at least one alcohol solvent has a boiling point at least or greater than about 150 °C, 175 °C or 200 °C at a pressure of about 1 atm.
• the at least one alcohol solvent having a boiling point greater than the boiling point of water allows for an aqueous mixture comprising a biological material to be mixed with at least one alcohol solvent and for water to be selectively removed (e.g., by evaporation) from the resulting aqueous organic mixture without substantial loss of the at least one alcohol solvent.
• the at least one alcohol solvent has a dynamic (or absolute) viscosity of no more than about 1500, 1000, 500, 400, 300, 200, 100, 50 or 25 centipoise (cP) or mPa-s at ambient temperature. In certain embodiments, the at least one alcohol solvent has a dynamic (or absolute) viscosity of no more than about 1000, 500, 200, 100 or 50 cP or mPa-s at ambient temperature. A lower dynamic (or absolute) viscosity of the at least one alcohol solvent allows for more facile handling of the composition comprising the biological material and the at least one alcohol solvent (e.g., using a pipette or other means of transferring the fluid composition).
• the linear and branched C 2 -C 6 acyclic alcohols having one or more hydroxyl groups and optionally comprising one or more halide atoms are linear and branched C 2 -C 5 acyclic alcohols having one hydroxyl group and optionally comprising one or more halide atoms.
• Non-limiting examples of linear and branched C 2 -C 5 acyclic alcohols having one hydroxyl group and optionally comprising one or more halide atoms include 2-chloroethanol, 2,2-dichloroethanol, 1 - butanol, 1 -pentanol, 2-methylbutan-l -ol, 3-methylbutan-l -ol, 2,2-dimethylpropan-l -ol, 2-pentanol, 3- methylbutan-2-ol, and 3-pentanol.
• the linear and branched C 2 -C 6 acyclic alcohols are linear and branched C2-C6 acyclic alcohols having two or more (e.g., two or three) hydroxyl groups and optionally comprising one or more halide atoms.
• Non-limiting examples of linear and branched C 2 - Ce acyclic alcohols having two or more (e.g., two or three) hydroxyl groups and optionally comprising one or more halide atoms include 1 ,2-ethanediol (ethylene glycol), 1 ,2-propanediol (propylene glycol), 1 ,3 -propanediol, 1 ,2,3-propanetriol (glycerol), 1 ,2-butanediol, 1 ,3-butanediol, 1 ,4-butanediol, 2,3-butanediol, 1 ,2,3-butanetriol, 1 ,2,4-butanetriol, 1 ,2-pentanediol, 1 ,3-pentanediol, 1 ,4-pentanediol, 1 ,5-pentanediol, 2,3-pentanedio
• the linear and branched C 2 -C 6 acyclic alcohols are linear and branched C 2 -C 5 acyclic alcohols having two or more (e.g., two or three) hydroxyl groups and optionally comprising one or more halide atoms.
• the at least one alcohol solvent comprises ethylene glycol, 1 ,2-propanediol, 1 ,3-propanediol, glycerol, 1 ,2-butanediol, 1,3- butanediol, 1 ,4-butanediol, 2,3-butanediol, 1 ,2,4-butanetriol or 1 ,5-pentanediol, or any combination thereof.
• the at least one alcohol solvent comprises ethylene glycol, 1 ,3- propanediol, glycerol or 1 ,2-butanediol, or any combination thereof.
• the C 3 -C 6 cyclic alcohols having one or more hydroxyl groups and optionally comprising one or more halide atoms are C4 or C 5 cyclic alcohols having one or more hydroxyl groups and four or five ring carbon atoms and optionally comprising one or more halide atoms.
• the at least one alcohol solvent comprises cyclobutanol or cyclopentanol, or both.
• the at least one alcohol solvent comprises two or more alcohol solvents.
• the at least one alcohol solvent comprises two or more alcohol solvents selected from the group consisting of ethylene glycol, 1 ,2-propanediol, 1 ,3-propanediol, glycerol, 1 ,2-butanediol, 1 ,3-butanediol, 1 ,4-butanediol, 2,3-butanediol, 1 ,2,4-butanetriol, and 1 ,5- pentanediol.
• the at least one alcohol solvent comprises: (a) ethylene glycol and glycerol; or (b) ethylene glycol and 1 ,2-butanediol; or (c) glycerol and 1 ,2-butanediol.
• the at least one alcohol solvent comprises glycerol and an additional alcohol solvent that can be any alcohol solvent described herein.
• the at least one alcohol solvent comprises glycerol and an additional alcohol solvent selected from the group consisting of ethylene glycol, 1 ,2-propanediol, 1,3-propanediol, 1 ,2-butanediol, 1,3-butanediol, 1,4- butanediol, 2,3-butanediol, 1,2,3-butanetriol, 1 ,2,4-butanetriol, 1 ,2-pentanediol, 1,3-pentanediol, 1,4- pentanediol, 1,5-pentanediol, 2,3-pentanediol, 2,4-pentanediol, 1,2,3-pentanetriol, 1 ,2,4-pentanetriol, 1,2,5-pentanetriol, 1,3,4-pentanetriol, 1,3,5-pentanetriol, 2,3,4-pentanetriol
• the composition comprises an enzyme.
• the enzyme mediates a nucleic acid reaction.
• the enzyme that mediates a nucleic acid reaction comprises a topoisomerase, a helicase, a DNA polymerase, a reverse transcriptase, an RNA polymerase, a DNA ligase, an RNA ligase, a DNA repair enzyme, an RNA repair enzyme, an endonuclease, an exonuclease, a deoxyribonuclease (DNase), a ribonuclease (RNase), a transposase, a restriction enzyme or a nicking enzyme, or any combination thereof.
• DNase deoxyribonuclease
• RNase ribonuclease
• the enzyme that mediates a nucleic acid reaction comprises a DNA polymerase, a reverse transcriptase or an RNA polymerase, or any combination thereof.
• the enzyme that mediates a nucleic acid reaction comprises a DNA polymerase (e.g., a heat-stable DNA polymerase, such as a Taq polymerase) used in PCR or a reverse transcriptase used in PCR, or both.
• PCR Polymerase chain reaction
• PCR includes standard PCR and variations thereof, such as allele-specific PCR, assembly PCR, asymmetric PCR, dial-out PCR, hot-start PCR, intersequence- specific PCR, inverse PCR, isothermal PCR (e.g., helicase-dependent amplification and PAN-AC), ligation-mediated PCR, methylation-specific PCR, mini-primer PCR, multiplex ligation-dependent probe amplification, multiplex PCR, nested PCR, overlap-extension PCR, picotiter PCR, quantitative PCR, real-time PCR, restriction fragment length polymorphism PCR, reverse transcription PCR (RT- PCR), single-cell PCR, solid-phase PCR (e.g., standard solid-phase PCR, enhanced solid-phase PCR, bridge PCR, and polony amplification), thermal asymmetric interlaced PCR, touchdown (step-down) PCR, and universal fast walking PCR.
• the composition comprises a polynucleotide used in a nucleic acid reaction.
• the polynucleotide comprises at least one primer used in PCR or reverse transcription.
• the at least one primer used in PCR or reverse transcription comprises at least one pair of a forward primer and a reverse primer for amplifying at least one nucleic acid (e.g., genetic) locus, or at least one reverse transcription primer for reverse transcribing at least one polyribonucleotide, or both.
• the at least one pair of forward primer and reverse primer is labeled with a dye (e.g., a fluorescent dye) or the at least one reverse transcription primer is labeled with a dye (e.g., a fluorescent dye), or both.
• the composition comprises a plurality of different pairs of forward and reverse primers for amplifying a plurality of different short tandem repeat (STR) loci utilized in a forensic database, such as the Combined DNA Index System (CODIS) recommended by the Federal Bureau of Investigation (FBI).
• CODIS Combined DNA Index System
• CODIS presently utilizes 13 STR loci dubbed CSF1PO, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21 S11, FGA, TH01, TPOX and vWA.
• the composition comprises at least 5 or at least 10, or 13, different pairs of forward and reverse primers for amplifying at least 5 or at least 10, or all 13, CODIS STR loci.
• the composition further comprises at least one pair of forward and reverse primers for amplifying at least one other STR locus useful for human identification, such as Penta D and Penta E.
• the composition further comprises at least one pair of forward and reverse primers for amplifying at least one nucleic acid (e.g., genetic) locus useful for sex determination, such as amelogenin (AMEL).
• at least one nucleic acid e.g., genetic locus useful for sex determination, such as amelogenin (AMEL).
• the composition comprises 16 different pairs of forward and reverse primers for amplifying all 13 CODIS STR loci, Penta D, Penta E and amelogenin.
• each of the 16 different pairs of forward and reverse primers is labeled with a dye (e.g., a fluorescent dye), which may be the same as or different from the dyes used to label the other pairs of forward and reverse primers (e.g., three, four or more spectrally resolvable fluorescent dyes can be used to label the 16 different pairs of forward and reverse primers).
• a dye e.g., a fluorescent dye
• the dyes used to label the other pairs of forward and reverse primers e.g., three, four or more spectrally resolvable fluorescent dyes can be used to label the 16 different pairs of forward and reverse primers.
• the composition comprises one or more reagents for performing PCR, wherein the reagents for performing PCR comprise a DNA polymerase and at least one pair of a forward primer and a reverse primer for amplifying at least one nucleic acid (e.g., genetic) locus, and the at least one pair of forward primer and reverse primer optionally is labeled with a dye (e.g., a fluorescent dye).
• a dye e.g., a fluorescent dye
• the at least one pair of forward and reverse primers comprises at least 5 or 10 different pairs of forward and reverse primers for amplifying at least 5 or 10 different STR loci utilized in a forensic database (e.g., CODIS), wherein each of the at least 5 or 10 different pairs of forward and reverse primers optionally is labeled with a dye (e.g., a fluorescent dye).
• a dye e.g., a fluorescent dye
• the at least one pair of forward and reverse primers comprises 16 different pairs of forward and reverse primers for amplifying all 13 CODIS STR loci, Penta D, Penta E and amelogenin.
• each of the 16 different pairs of forward and reverse primers is labeled with a dye (e.g., a fluorescent dye), which may be the same as or different from the dyes used to label the other pairs of forward and reverse primers (e.g., three, four or more spectrally resolvable fluorescent dyes can be used to label the 16 different pairs of forward and reverse primers).
• the DNA polymerase comprises a DNA polymerase that is stable at elevated temperature (e.g., at about 60 °C, 70 °C, 80 °C, 90 °C or higher), such as a Taq polymerase.
• the reagents for performing PCR further comprise deoxyribonucleotide triphosphates.
• the reagents for performing PCR further comprise a buffer or a metal salt (e.g., an M +1 or M +2 salt, such as magnesium chloride), or both.
• the composition comprises the DNA polymerase, the at least one pair of forward and reverse primers, deoxyribonucleotide triphosphates, and optionally a buffer and/or a metal salt (e.g., an M +1 or M +2 salt, such as magnesium chloride) for performing PCR.
• the composition comprises the DNA polymerase and no primer, where the composition can further comprise deoxyribonucleotide triphosphates and optionally a buffer and/or a metal salt (e.g., an M +1 or M +2 salt, such as magnesium chloride).
• the composition comprises the at least one pair of forward and reverse primers and no DNA polymerase, where the composition can further comprise deoxyribonucleotide triphosphates and optionally a buffer and/or a metal salt (e.g., an M +1 or M +2 salt, such as magnesium chloride).
• a kit containing reagents for performing PCR can contain, for example:
• composition comprising the DNA polymerase, the at least one pair of forward and reverse primers, deoxyribonucleotide triphosphates, and optionally a buffer and/or a metal salt; or
• composition comprising the DNA polymerase and no primer, which can further comprise deoxyribonucleotide triphosphates and optionally a buffer and/or a metal salt;
• a separate composition comprising the at least one pair of forward and reverse primers and no DNA polymerase, which can further comprise deoxyribonucleotide triphosphates and optionally a buffer and/or a metal salt.
• the composition comprises one or more PCR reagents for amplifying at least one nucleic acid (e.g., genetic) sequence of the DNA of a microbe or a pathogen, such as the DNA of any bacterium described herein, the DNA of any fungus described herein, and the DNA of any DNA virus described herein.
• a kit can contain a composition comprising all of the PCR reagents for amplifying at least one nucleic acid (e.g., genetic) sequence of the DNA of a microbe or a pathogen, or two or more compositions that in total comprise all of the PCR reagents.
• the composition comprises one or more reagents for performing reverse transcription, wherein the reagents for performing reverse transcription comprise a reverse transcriptase and at least one reverse transcription primer for reverse transcribing at least one polyribonucleotide, and the at least one reverse transcription primer optionally is labeled with a dye (e.g., a fluorescent dye).
• the reagents for performing reverse transcription further comprise deoxyribonucleotide triphosphates.
• the reagents for performing reverse transcription further comprise a buffer or a metal salt (e.g., an M +1 or M +2 salt, such as magnesium chloride), or both.
• the composition comprises the reverse transcriptase, the at least one reverse transcription primer, deoxyribonucleotide triphosphates and optionally a buffer and/or a metal salt (e.g., an M +1 or M +2 salt, such as magnesium chloride) for performing reverse transcription.
• a metal salt e.g., an M +1 or M +2 salt, such as magnesium chloride
• the composition comprises the reverse transcriptase and no reverse transcriptase
• compositions can further comprise deoxyribonucleotide triphosphates and optionally a buffer and/or a metal salt (e.g., an M +1 or M +2 salt, such as magnesium chloride).
• the composition comprises the at least one reverse transcription primer and no reverse transcriptase, where the composition can further comprise deoxyribonucleotide triphosphates and optionally a buffer and/or a metal salt (e.g., an M +1 or M +2 salt, such as magnesium chloride).
• a kit containing reagents for performing reverse transcription can contain, for example: 1) a
• composition comprising the reverse transcriptase, the at least one reverse transcription primer, deoxyribonucleotide triphosphates and optionally a buffer and/or a metal salt; or 2) (i) a composition comprising the reverse transcriptase and no reverse transcription primer, which can further comprise deoxyribonucleotide triphosphates and optionally a buffer and/or a metal salt; and (ii) a separate composition comprising the at least one reverse transcription primer and no reverse transcriptase, which can further comprise deoxyribonucleotide triphosphates and optionally a buffer and/or a metal salt.
• the composition comprises one or more reagents for performing reverse transcription PCR (RT-PCR), wherein: the reagents for performing RT-PCR comprise a reverse transcriptase, a DNA polymerase, at least one reverse transcription primer for reverse transcribing at least one polyribonucleotide to produce at least one polydeoxyribonucleotide complementary to the at least one polyribonucleotide, and at least one pair of a forward primer and a reverse primer for amplifying the at least one complementary polydeoxyribonucleotide; and the at least one reverse transcription primer optionally is labeled with a dye (e.g., a fluorescent dye) and the at least one pair of forward primer and reverse primer optionally is labeled with a dye (e.g., a fluorescent dye).
• a dye e.g., a fluorescent dye
• the DNA polymerase comprises a DNA polymerase that is stable at elevated temperature (e.g., at about 60 °C, 70 °C, 80 °C, 90 °C or higher), such as a Taq polymerase.
• the reagents for performing RT-PCR further comprise
• the reagents for performing RT-PCR further comprise a buffer or a metal salt (e.g., an M +1 or M +2 salt, such as magnesium chloride), or both.
• a buffer or a metal salt e.g., an M +1 or M +2 salt, such as magnesium chloride
• the composition comprises the reverse transcriptase, the DNA polymerase, the at least one reverse transcription primer, the at least one pair of forward and reverse primers, deoxyribonucleotide triphosphates, and optionally a buffer and/or a metal salt (e.g., an M +1 or M +2 salt, such as magnesium chloride) for performing RT-PCR.
• the composition comprises the reverse transcriptase, the DNA polymerase, no reverse transcription primer, and no pair of forward and reverse primers, where the composition can further comprise deoxyribonucleotide triphosphates and optionally a buffer and/or a metal salt (e.g., an M +1 or M +2 salt, such as magnesium chloride).
• the composition comprises the at least one reverse transcription primer, the at least one pair of forward and reverse primers, no reverse transcriptase, and no DNA polymerase , where the composition can further comprise
• a kit containing reagents for performing RT-PCR can contain, for example: 1) a composition comprising the reverse transcriptase, the DNA polymerase, the at least one reverse transcription primer, the at least one pair of forward and reverse primers, deoxyribonucleotide triphosphates, and optionally a buffer and/or a metal salt; or 2) (i) a composition comprising the reverse transcriptase, the DNA polymerase, no reverse transcription primer, and no pair of forward and reverse primers, which can further comprise deoxyribonucleotide triphosphates and optionally a buffer and/or a metal salt; and (ii) a separate composition comprising the at least one reverse transcription primer, the at least one pair of forward and reverse primers, no reverse transcriptase, and no DNA polymerase , which can further comprise de
• the composition comprises one or more RT-PCR reagents for amplifying at least one polydeoxyribonucleotide complementary to at least one nucleic acid (e.g., genetic) sequence of the RNA of an RNA virus, such as the RNA of any RNA virus described herein.
• a kit can contain a composition comprising all of the RT-PCR reagents for amplifying at least one polydeoxyribonucleotide complementary to at least one nucleic acid (e.g., genetic) sequence of the RNA of an RNA virus, or two or more compositions that in total comprise all of the RT-PCR reagents.
• the composition comprises reagents for performing transcription, wherein the reagents for performing transcription comprise an RNA polymerase.
• the reagents for performing transcription further comprise ribonucleotide triphosphates.
• the reagents for performing transcription further comprise a buffer or a metal salt (e.g., an M +1 or M +2 salt, such as magnesium chloride), or both.
• the composition comprises a polypeptide that regulates (e.g., agonizes or antagonizes/inhibits) an enzyme.
• polypeptides that regulate enzymes include polypeptides that inhibit proteases, such as the protease inhibitors described herein.
• the composition comprises an antibody.
• the antibody is used in an immunoassay.
• the immunoassay is an enzyme- linked immunosorbent assay (ELISA) or a sandwich immunoassay.
• the antibody used in an immunoassay comprises an unlinked antibody, an antibody bound to a solid substrate (e.g., a bead), an antibody conjugated to a detection protein or enzyme or a fragment thereof, or any combination thereof, wherein the antibody may or may not be labeled with a dye (e.g., a fluorescent dye, a chemiluminescent dye, or a phosphorescent dye).
• the composition comprises one or more reagents for performing an immunoassay, wherein the one or more reagents for performing an immunoassay comprise an antibody that has affinity for or is specific for a target antigen or analyte, the antibody is labeled with a dye (e.g., a fluorescent dye, a chemiluminescent dye, or a phosphorescent dye) or is conjugated to a detection protein or enzyme or a fragment thereof, and the antibody optionally is bound to a solid substrate (e.g., a bead).
• a dye e.g., a fluorescent dye, a chemiluminescent dye, or a phosphorescent dye
• the composition comprises one or more reagents for performing a sandwich immunoassay, wherein: the reagents for performing a sandwich immunoassay comprise a first antibody that has affinity for or is specific for a target antigen or analyte and a second antibody that has affinity for or is specific for the target antigen or analyte; the second antibody is labeled with a dye (e.g., a fluorescent dye, a chemiluminescent dye, or a phosphorescent dye) or is conjugated to a detection protein or enzyme or a fragment thereof; and the first antibody optionally is bound to a solid substrate (e.g., a bead).
• a dye e.g., a fluorescent dye, a chemiluminescent dye, or a phosphorescent dye
• the composition comprises the first antibody and the second antibody. In other embodiments, the composition comprises the first antibody and not the second antibody. In yet other embodiments, the composition comprises the second antibody and not the first antibody.
• a kit containing reagents for performing a sandwich immunoassay can contain, for example: 1) a composition comprising the first antibody and the second antibody; or 2) (i) a composition comprising the first antibody and not the second antibody; and (ii) a separate composition comprising the second antibody and not the first antibody.
• the composition comprises one or more reagents for performing an enzyme-linked immunosorbent assay (ELISA), wherein the reagents for performing ELISA comprise: a detection antibody that has affinity for or is specific for a target antigen or analyte and is conjugated to a detection enzyme or a fragment thereof; or a first antibody that has affinity for or is specific for a target antigen or analyte, and a second antibody that has affinity for or is specific for the first antibody and is conjugated to a detection enzyme or a fragment thereof.
• ELISA enzyme-linked immunosorbent assay
• the composition comprises the detection antibody. In other embodiments, the composition comprises the first antibody and the second antibody. In yet other embodiments, the composition comprises the first antibody and not the second antibody. In still other embodiments, the composition comprises the second antibody and not the first antibody.
• a kit containing reagents for performing an ELISA can contain, for example: 1) a composition comprising the detection antibody; or 2) a composition comprising the first antibody and the second antibody; or 3) (i) a composition comprising the first antibody and not the second antibody; and (ii) a separate composition comprising the second antibody and not the first antibody.
• the detection protein or enzyme or a fragment thereof that is conjugated to an antibody used in an immunoassay can be any protein or enzyme or any fragment thereof that is suitable for detection.
• the detection protein or enzyme or a fragment thereof that is conjugated to an antibody used in an immunoassay is selected from the group consisting of: a) phycobiliproteins, phycoerythrins, B-phycoerythrin, R-phycoerythrin, and fragments and conjugates thereof; b) streptavidin, avidin, deglycosylated avidin, and fragments and conjugates thereof; c) peroxidases, horseradish peroxidase, and fragments and conjugates thereof; and d) phosphatases, alkaline phosphatase, and fragments and conjugates thereof.
• the composition comprises a protein or enzyme useful for detection, where the protein or enzyme may or may not be conjugated to an antibody.
• proteins and enzymes useful for detection include: a) phycobiliproteins, phycoerythrins, B-phycoerythrin, R-phycoerythrin, and conjugates thereof (e.g., phycoerythrin-streptavidin conjugates, phycoerythrin-alkaline phosphatase conjugates, and phycoerythrin-antibody conjugates); b) streptavidin, avidin, deglycosylated avidin, and conjugates thereof (e.g., streptavidin-horse radish peroxidase conjugates and streptavidin-antibody conjugates); c) peroxidases, horseradish peroxidase, and conjugates thereof (e.g., horseradish peroxidase-antibody conjugates); and d)
• the composition comprises a polypeptide aptamer that binds specifically to a target ligand (e.g., a small molecule, a protein, a nucleic acid, a cell, a tissue or an organism).
• a target ligand e.g., a small molecule, a protein, a nucleic acid, a cell, a tissue or an organism.
• the polypeptide aptamer comprises a variable domain or loop (e.g., a domain or loop containing about 10 to about 20 amino acids) attached at both ends to a polypeptide scaffold (e.g., a protein scaffold, such as thioredoxin A).
• composition can also contain other kinds of polypeptides, including without limitation receptors (e.g., peripheral membrane proteins, transmembrane proteins and nuclear receptors), polypeptide ligands (e.g., polypeptide ligands of antibodies), regulatory factors, hormones, cytokines (e.g., interferons and inter leukins), structural proteins (e.g., collagen and elastin), and toxins.
• receptors e.g., peripheral membrane proteins, transmembrane proteins and nuclear receptors
• polypeptide ligands e.g., polypeptide ligands of antibodies
• regulatory factors e.g., hormones, cytokines (e.g., interferons and inter leukins), structural proteins (e.g., collagen and elastin), and toxins.
• cytokines e.g., interferons and inter leukins
• structural proteins e.g., collagen and elastin
• Non-limiting examples of hormones include adrenocorticotropic hormone, angiotensin II, antidiuretic hormone (vasopressin), basic fibroblast growth factor-2, cholecystokinin, colony- stimulating factors (e.g., granulocyte colony-stimulating factor), gastrin, growth hormone, insulin, leptin, atrial natriuretic peptide, brain natriuretic peptide, C-type natriuretic peptide, oxytocin, parathyroid hormone -related protein, prolactin, and somatostatin.
• adrenocorticotropic hormone angiotensin II, antidiuretic hormone (vasopressin), basic fibroblast growth factor-2, cholecystokinin, colony- stimulating factors (e.g., granulocyte colony-stimulating factor), gastrin, growth hormone, insulin, leptin, atrial natriuretic peptide,
• toxins include without limitation cyanotoxins, cytotoxins, exotoxins (e.g., botulinum toxin and Corynebacterium diphtheriae exotoxin), hemotoxins, hepatotoxins (e.g., amatoxins and phallotoxins), mycotoxins, necrotoxins, neurotoxins (e.g., bungarotoxins, chlorotoxin, conotoxins and tetanus toxin), plant toxins (e.g., ricin), insect toxins (e.g., apitoxin), and snake toxins [e.g., cardiotoxins, myotoxins, neurotoxins (such as alpha-neurotoxins, beta-neurotoxins and dendrotoxins), sarafotoxins, hydrolases (such as
• phosphodiesterases and phospholipases phospholipases
• lyases phosphodiesterases and phospholipases
• oxydoreductases such as L-amino acid oxidases
• transferases hemorrhagins, hyaluronidases, thrombin-like pro-coagulants, and kallikrein-like serine proteases.
• composition can preserve polypeptide therapeutics (e.g., hormone therapeutics, cytokine therapeutics, antibody therapeutics, fusion protein therapeutics,
• polypeptide therapeutics e.g., hormone therapeutics, cytokine therapeutics, antibody therapeutics, fusion protein therapeutics,
• Hormone therapeutics include without limitation erythropoietin, growth hormone, insulin, and other hormones described herein.
• Cytokine therapeutics include without limitation interferons [e.g., interferon alpha (including interferon alpha-2a and interferon alpha-2b), interferon beta (including interferon beta-la and interferon beta-lb), and derivatives thereof (including interferons derivatived with polyethylene glycol (PEG))] and interleukins (e.g., interleukin 2 and interleukin 12).
• interferons e.g., interferon alpha (including interferon alpha-2a and interferon alpha-2b), interferon beta (including interferon beta-la and interferon beta-lb), and derivatives thereof (including interferons derivatived with polyethylene glycol (PEG))
• interleukins e.g., interleukin 2 and interleukin 12
• Non- limiting examples of antibody therapeutics include adalimumab, bevacizumab, infliximab, trastuzumab, and ustekinumab.
• Fusion protein therapeutics include without limitation abatacept, alefacept, denileukin diftitox, and etanercept.
• antithrombotics include anti-platelet agents (e.g., abciximab), anticoagulants (e.g., antithrombin, batroxobin, hementin, hirudin, lepirudin and bivalirudin), and thrombolytics [e.g., tissue plasminogen activators (including alteplase, reteplase and tenecteplase), anistreplase, streptokinase and urokinase].
• tissue plasminogen activators including alteplase, reteplase and tenecteplase
• anistreplase streptokinase and urokinase
• toxin therapeutics include without limitation botulinum toxin, chlorotoxin, and toxoids used as vaccines (e.g., against botulism, diphtheria and tetanus).
• Non- limiting examples of vaccines include vaccines against botulism, bubonic plague, chicken pox, cholera, diphtheria, hepatitis, influenza, measles, mumps, polio, rabies, rubella, small pox, tetanus, tuberculosis, typhoid, and yellow fever.
• the composition contains a pharmaceutical formulation comprising a polypeptide and optionally one or more other substances as described herein.
• a pharmaceutical formulation comprising a polypeptide and optionally one or more other substances as described herein.
• the concentration of the polypeptide in the composition is at least about 10 mg/mL, 50 mg/mL, 100 mg/mL, 200 mg/mL, 300 mg/mL, 400 mg/mL, 500 mg/mL or 1 g/mL, or about 100- 1000 mg/mL, 100-500 mg/mL or 500-1000 mg/mL. In some embodiments, the concentration of the polypeptide in the composition is at least about 100 mg/mL, or about 100-1000 mg/mL.
• the composition comprises a catalytic polynucleotide.
• the catalytic polynucleotide is a natural or synthetic ribozyme (or RNA enzyme or catalytic RNA).
• natural ribozymes include peptidyl transferase 23 S rRNA, RNase P, Group I introns, Group II introns, GIR1 branching ribozyme, leadzyme, hairpin ribozyme, hammerhead ribozyme, HDV ribozyme, mammalian CPEB3 ribozyme, VS ribozyme, glmS ribozyme, and CoTC ribozyme.
• RNA polymerases e.g., Round 18 RNA polymerase ribozyme and variants thereof, such as B6.61 ribozyme and tC19Z ribozyme
• ribozymes produced from RNA ligases e.g., the catalytic polynucleotide is a deoxyribozyme (or DNA enzyme or catalytic DNA), including deoxyribozymes that catalyze DNA phosphorylation, DNA adenylation, DNA deglycosylation, DNA cleavage, thymine dimer photoreversion, and porphyrin metalation.
• the composition comprises a polynucleotide that binds specifically to a target ligand (e.g., a small molecule, a protein, a nucleic acid, a cell, a tissue or an organism).
• a target ligand e.g., a small molecule, a protein, a nucleic acid, a cell, a tissue or an organism.
• the polynucleotide that binds specifically to a target ligand is a natural or synthetic nucleic acid aptamer (e.g., DNA aptamer, RNA aptamer or XNA aptamer).
• Non- limiting examples of nucleic acid aptamers include DNA aptamers and RNA aptamers that bind dopamine, hemin, HIV trans-acting responsive element, interferons (e.g., interferon-gamma), lysozymes, mycotoxins, thrombin, and vascular endothelial growth factor (VEGF).
• DNA aptamers and RNA aptamers that bind dopamine, hemin, HIV trans-acting responsive element, interferons (e.g., interferon-gamma), lysozymes, mycotoxins, thrombin, and vascular endothelial growth factor (VEGF).
• interferons e.g., interferon-gamma
• lysozymes e.g., mycotoxins
• thrombin vascular endothelial growth factor
• the composition comprises a biological sample.
• the biological sample can be, e.g., a clinical sample, a surgical sample, a laboratory sample, a research sample, a forensic sample, a veterinary sample, an environmental sample, an agricultural sample, or an industrial sample.
• the biological sample can be re-hydrated by addition of water or an aqueous solution (e.g., an aqueous buffer) for analysis, if desired.
• the biological sample comprises whole or fractionated animal (e.g., mammalian, such as human) blood.
• the biological sample comprises whole or fractionated animal (e.g., mammalian, such as human) plasma.
• the biological sample comprises whole or fractionated animal (e.g., mammalian, such as human) serum.
• the biological sample comprises cells.
• a cell can be, e.g., a eukaryotic or prokaryotic cell from any single-celled or multi-celled organism, and can be of any type.
• the biological sample comprises animal cells, mammalian cells, human cells, plant cells, microbial cells, pathogenic cells, bacterial cells, fungal cells, protozoan cells or viral particles, or any combination thereof, or lysates or extracts thereof.
• the cells or viral particles can be dissolved or suspended in a natural fluid or a laboratory culture medium (e.g., Dulbecco's phosphate buffered saline with 2% fetal bovine serum, Eagle's minimum essential medium (EMEM), Dulbecco's modified Eagle's medium (DMEM), or the allantoic fluid of embryonated chicken eggs) and then transferred to the substantially water- free fluid medium containing the at least one alcohol solvent (or the at least one ionic organic solvent in other embodiments of a composition comprising a biological material in a substantially water- free fluid medium, as described below).
• EMEM Eagle's minimum essential medium
• DMEM Dulbecco's modified Eagle's medium
• the biological sample comprises a bacterium.
• the bacterium can be non-pathogenic or pathogenic.
• bacteria include Bacillus (e.g., Bacillus anthracis, Bacillus cereus and Bacillus thuringiensis); Bordetella (e.g., Bordetella pertussis); Borrelia (e.g., Borrelia burgdorferi); Brucella (e.g., Brucella abortus, Brucella canis, Brucella melitensis, and Brucella suis); Campylobacter (e.g., Campylobacter jejuni); Chlamydia and Chlamydophila (e.g., Chlamydia pneumoniae, Chlamydia trachomatis, and Chlamydophila psittaci); Clostridium (e.g., Clostridium botulinum, Clostridium difficile, Clostridium perfringen
• Corynebacterium e.g., Corynebacterium diphtheriae
• Enterococcus e.g., Enterococcus faecalis and Enterococcus faecium
• Escherichia e.g., Escherichia coli
• Francisella e.g., Francisella tularensis
• Haemophilus e.g., Haemophilus influenzae
• Helicobacter e.g., Helicobacter pylori
• Legionella e.g., Legionella pneumophila
• Leptospira e.g., Leptospira interrogans
• Listeria e.g., Listeria monocytogenes
• Mycobacterium e.g., Mycobacterium leprae, Mycobacterium tuberculosis, and Mycobacterium ulcerans
• Mycoplasma e.g., Mycoplasma pneumoniae
• Neisseria
• the biological sample comprises a fungus.
• the fungus can be nonpathogenic or pathogenic.
• examples of fungi include without limitation Aspergillus (e.g., Aspergillus clavatus, Aspergillus fumigatus, and Aspergillus flavus); Blastomyces (e.g., Blastomyces
• Candida e.g., Candida albicans
• Coccidioides e.g., Coccidioides immitis
• Cryptococcus e.g., Cryptococcus albidus, Cryptococcus gattii,
• Cryptococcus laurentii, and Cryptococcus neoformans Fusarium (e.g., Fusarium graminearum, Fusarium oxysporum, Fusarium proliferatum, Fusarium solani complex, and Fusarium
• Histoplasma e.g., Histoplasma capsulatum
• Pneumocystis e.g., Pneumocystis jirovecii (or Pneumocystis carinii)]
• Stachybotrys e.g., Stachybotrys chartarum
• Trichosporon e.g., Trichosporon asahii, Trichosporon asteroides, Trichosporon cutaneum, Trichosporon dermatis, Trichosporon dohaense, Trichosporon inkin, Trichosporon loubieri, Trichosporon mucoides and Trichosporon ovoides
• Zygomycetes e.g., Rhizopus stolonifer
• the biological sample comprises a protozoan.
• the protozoan can be non-pathogenic or pathogenic.
• Examples of protozoa include without limitation Balantidium (e.g., Balantidium coli); Cryptosporidium (e.g., Cryptosporidium canis, Cryptosporidium felis, Cryptosporidium hominis, Cryptosporidium meleagridis, Cryptosporidium muris and
• Entamoeba e.g., Entamoeba dispar and Entamoeba histolytica
• Giardia e.g., Giardia lamblia and Giardia muris
• Leishmania e.g., Leishmania braziliensis, Leishmania infantum and Leishmania major
• Naegleria e.g., Naegleria fowleri
• Plasmodia e.g., Plasmodia falciparum, Plasmodia knowlesi, Plasmodia malariae and Plasmodia vivax
• Toxoplasma e.g., Toxoplasma gondii
• Trichomonas e.g., Trichomonas vaginalis
• Trypanosoma e.g.,
• the biological sample comprises a viras.
• the viras can be a DNA viras or an RNA virus.
• DNA viruses include adenoviruses [including Atadenovirus (e.g., ovine adenovirus D), Aviadenovirus (e.g., fowl adenovirus A), Ichtadenovirus (e.g., sturgeon adenovirus A), Mastadenovirus (e.g., human adenovirus C and AD-36), and
• Siadenovirus e.g., frog adenovirus
• hepadnavirases including Orthohepadnavirus (e.g., hepatitis B virus) and Avihepadnavirus (e.g., duck hepatitis B virus)
• herpesviruses include human herpesviruses (e.g., herpes simplex virus-1 , herpes simplex viras-2, Varicella zoster viras, Epstein- Barr viras, cytomegalovirus, roseoloviras, herpes lymphotropic virus, pityriasis rosea viras, and Kaposi's sarcoma-associated herpesvirus) and zoonotic herpesviruses (e.g., cercopithecine herpesvirus-1 and murine gammaherpesviras-68)]
• papillomaviruses e.g., human herpes
• polyomaviras and human polyomavirus 9].
• Non-limiting examples of RNA viruses include coronaviruses [including human coronaviruses (e.g., SARS coronavirus)]; flavivirases [including Flavivirus (e.g., yellow fever viras, West Nile viras, and dengue fever viras), Hepacivirus (e.g., hepatitis C virus), Hepatitis G Virus (e.g., the GB agent and hepatitis G viras), and Pestivirus (e.g., bovine viral diarrhea viras, classical swine fever virus, and hog cholera virus)]; orthomyxoviruses [including Influenzavirus A (e.g., influenza A virus), Influenzavirus B (e.g., influenza B viras), Influenzavirus C (e.g., influenza C virus), Isavirus (e.g., infectious salmon anemia viras), and Thogotovirus (e.g., Dhori virus
• paramyxoviruses [including Aquaparamyxovirus (e.g., Atlantic salmon paramyxovirus and Pacific salmon paramyxovirus), Avulavirus (e.g., Newcastle disease viras), Ferlavirus (e.g., Fer-de-Lance virus), Henipavirus (e.g., hendravirus and nipahviras), Morbillivirus (e.g., measles viras, canine distemper viras, ovine rinderpest viras, phocine distemper virus, and rinderpest virus), Respirovirus (e.g., human parainfluenza viruses 1 and 3, and Sendai viras), Rubulavirus (e.g., mumps viras, human parainfluenza viruses 2 and 4, Menangle viras, simian parainfluenza virus 5, Tioman viras, and Tuhokoviruses 1 , 2 and 3),
• HIV immunodeficiency viruses
• simian immunodeficiency viruses simian immunodeficiency viruses
• feline immunodeficiency viruses simian foamy virus
• rhabdoviruses include Cytorhabdovirus (e.g., lettuce necrotic yellows virus), Dichorhabdovirus (e.g., orchid fleck virus), Ephemerovirus (e.g., bovine ephemeral fever virus), Lyssavirus (e.g., rabies virus), Novirhabdovirus (e.g., infectious hematopoietic necrosis virus), Nudeorhabdovirus (e.g., potato yellow dwarf virus), and Vesiculovirus (e.g., vesicular stomatitis Indiana virus)]; and togaviruses [including Rubivirus (e.g., rubella virus) and Alphavirus (e.g., Chikungunya virus, Eastern equine ence
• the composition containing the biological material further comprises one or more substances selected from the group consisting of reducing agents, antioxidants, free radical scavengers, oxygen radical scavengers, hydroxyl radical scavengers, singlet oxygen quenchers, hydroperoxide-removing agents, protease inhibitors, nuclease inhibitors, ribonuclease (RNase) inhibitors, deoxyribonuclease (DNase) inhibitors, metal chelators, preservatives, antimicrobials, buffers (or buffering agents), detergents, and chaotropes.
• reducing agents antioxidants, free radical scavengers, oxygen radical scavengers, hydroxyl radical scavengers, singlet oxygen quenchers, hydroperoxide-removing agents, protease inhibitors, nuclease inhibitors, ribonuclease (RNase) inhibitors, deoxyribonuclease (DNase) inhibitors, metal chelators, pre
• the composition can comprise the one or more substances in appropriate amounts to enhance, e.g., the stability and/or the solubility of the biological material in the substantially water-free fluid medium.
• a substance can have one or more functions or properties.
• a substance can be a reducing agent, an antioxidant, a free radical scavenger, an oxygen radical scavenger, a hydroxyl radical scavenger or a singlet oxygen quencher, or any combination thereof.
• a substance can be a metal chelator, a DNase inhibitor or an anti-microbial, or any combination thereof.
• Examples of reducing agents, antioxidants, and free radical scavengers include without limitation cysteine, dithionite, dithioerythritol, dithiothreitol (DTT), dysteine, 2-mercaptoethanol, mercaptoethylene, bisulfite, sodium metabisulfite, pyrosulfite, pentaerythritol, thioglycolic acid, urea, uric acid, vitamin C, vitamin E, superoxide dismutases, and analogs, derivatives and salts thereof.
• cysteine dithionite
• dithioerythritol dithiothreitol (DTT)
• DTT dithiothreitol
• 2-mercaptoethanol 2-mercaptoethanol
• mercaptoethylene bisulfite
• sodium metabisulfite sodium metabisulfite
• pyrosulfite pentaerythritol
• thioglycolic acid ure
• Non-limiting examples of oxygen radical scavengers include sugar alcohols (e.g., erythritol, mannitol, sorbitol, and xylitol), monosaccharides (e.g., hexoses, allose, altrose, fructose, fucose, fuculose, galactose, glucose, gulose, idose, mannose, rhamnose, sorbose, tagatose, talose, pentoses, arabinose, lyxose, ribose, deoxyribose, ribulose, xylose, xylulose, tetroses, erythrose, erythrulose, and threose), disaccharides (e.g., cellobiose, lactose, maltose, sucrose, and trehalose), complex sugars (e.g., trisaccharides, kes
• hydroxyl radical scavengers include without limitation azides (e.g., sodium azide), cysteine, dimethylsulfoxide, histidine, salicylic acid, salicylate, sugar alcohols (e.g., erythritol, mannitol, sorbitol, and xylitol), monosaccharides (e.g., those described herein), disaccharides (e.g., cellobiose, lactose, maltose, sucrose, and trehalose), complex sugars (e.g., those described herein), and analogs, derivatives and salts thereof.
• azides e.g., sodium azide
• cysteine dimethylsulfoxide
• histidine e.g., salicylic acid
• salicylate e.g., erythritol, mannitol, sorbitol, and xylitol
• monosaccharides e.g., those described
• Non-limiting examples of singlet oxygen quenchers include azides (e.g., sodium azide), ascorbic acid, ascorbate, alkyl imidazoles (e.g., carnosine, histamine, histidine, and imidazole 4-acetic acid), indoles (e.g., tryptophan and derivatives thereof, such as N-acetyl -5-methoxytryptamine, N- acetyl serotonin, and 6-methoxy-l,2,3,4-tetrahydro-beta-carboline), sulfur-containing amino acids (e.g., cysteine, S-allyl-cysteine, S-aminoethyl-cysteine, djenkolic acid, ethionine, methionine, N- formyl-methionine, lanthionine, and felinine), phenolic compounds (e.g., tyrosine and derivatives thereof), aromatic carboxylic acids (e.g
• hydroperoxide-removing agents include without limitation catalase, glutathione, peroxidases, glutathione peroxidases, pyruvate, and analogs, derivatives and salts thereof.
• Non-limiting examples of protease inhibitors include aspartic protease inhibitors, cysteine protease inhibitors, metalloprotease inhibitors, serine protease inhibitors, threonine protease inhibitors, trypsin inhibitors (e.g., avian egg white trypsin inhibitors, bovine trypsin inhibitors, lima bean trypsin inhibitors, and soybean trypsin inhibitors such as Kunitz trypsin inhibitor and Bowman- Birk inhibitor), Kunitz-type protease inhibitors, 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF) and salts thereof (e.g., HC1 salt), amastatin, antithrombin III, antipain, APMSF, aprotinin, bestatin, benzamidine, calpain inhibitors I and II, chymostatin, 3,4-dichloroisocoumarin, diisopropy
• Non-limiting examples of RNase inhibitors include mammalian ribonuclease inhibitor proteins [e.g., porcine ribonuclease inhibitor and human ribonuclease inhibitor (e.g., human placenta ribonuclease inhibitor and recombinant human ribonuclease inhibitor)], aurintricarboxylic acid (ATA) and salts thereof [e.g., triammonium aurintricarboxylate (aluminon)], adenosine 5 '-pyrophosphate, 2'- cytidine monophosphate free acid (2'-CMP), 5'-diphosphoadenosine 3'-phosphate (ppA-3'-p), 5'- diphosphoadenosine 2'-phosphate (ppA-2'-p), leucine, oligovinysulfonic acid, poly(aspartic acid), tyrosine-glutamic acid polymer, 5
• DNase inhibitors and metal chelators include without limitation
• aurintricarboxylic acid and salts thereof [e.g., triammonium aurintricarboxylate (aluminon)], boric acid, borate, citric acid, citrate, salicylic acid, salicylate, l,2-bis(o-aminophenoxy)ethane- ⁇ , ⁇ , ⁇ ', ⁇ '-tetraacetic acid (BAPTA), diethylene triamine pentaacetic acid (DTPA),
• EDTA ethylenediaminetetraacetic acid
• EGTA ethylene glycol tetraacetic acid
• GEDTA glycoletherdiaminetetraacetic acid
• HEDTA N-(2-hydroxyethyl)ethylenediamine-N,N',N'-triacetic acid
• NTA nitrilotriacetic acid
• 2,2'-bipyridine 2,2'-bipyridine
• o-phenanthroline triethanolamine, and analogs, derivatives and salts thereof.
• preservatives include without limitation azides (e.g., sodium azide), polyethylene glycol (PEG), and anti-microbials (e.g., anti-biotic, anti-fungal, anti-parasitic and antiviral agents).
• azides e.g., sodium azide
• PEG polyethylene glycol
• anti-microbials e.g., anti-biotic, anti-fungal, anti-parasitic and antiviral agents.
• Anti-microbials include without limitation beta-lactams, penicillins, semi-synthetic penicillins, mono-bactams, carboxypenems, aminoglycosides, glycopeptides, lincomycins, macrolides, allylamines, azoles, polyenes, tetraenes, sulfonamides, pyrimidines, thiocarbamates, benzoic acid compounds, rifamycins, tetracyclines, reverse transcriptase inhibitors, protease inhibitors, thymidine kinase inhibitors, glycoprotein synthesis inhibitors, sugar synthesis inhibitors, glucan synthesis inhibitors, structural protein synthesis inhibitors, viral maturation inhibitors, nucleoside analogs, polypeptides, and analogs, derivatives and salts thereof.
• antimicrobials include without limitation penicillin, cephalosporin, ampicillin, amoxycillin, aztreonam, clavulanic acid, imipenem, streptomycin, gentamycin, vancomycin, clindamycin, polymyxin, erythromycin, bacitracin, amphotericin, nystatin, rifampicin, tetracycline, chlortetracycline, doxycycline, chloramphenicol, ammolfme, butenafine, naftifine, terbinafme, ketoconazole, fluconazole, elubiol, econazole, econaxole, itraconazole, isoconazole, imidazole, miconazole, sulconazole, clotrimazole, enilconazole, oxiconazole, tioconazole, terconazole, butoconazole, thiabend
• the buffers or buffering agents provide buffering in a basic pH range (e.g., about pH 7 or 8 to 11, about pH 7 or 8 to 10, about pH 7 or 8 to 9, about pH 10-11, about pH 9-10, about pH 8-9, or about pH 7-8).
• a basic pH range e.g., about pH 7 or 8 to 11, about pH 7 or 8 to 10, about pH 7 or 8 to 9, about pH 10-11, about pH 9-10, about pH 8-9, or about pH 7-8).
• TES tris(hydroxymethyl)methyl] amino ⁇ ethanesulfonic acid
• CABS 3-amino-l -propanesulfonic acid
• CABS 3-(cyclohexylamino)-2-hydroxy-l- propanesulfonic acid
• EPPS N-(2-hydroxyethyl)piperazine-N'-(3-propanesulfonic acid)
• MOPS 3- (N-morpholino)propanesulfonic acid
• TAPS 3- ⁇ [tris(hydroxymethyl)methyl]amino ⁇ - propanesulfonic acid
• TPASO 4-(cyclohexylamino)-l -butanesulfonic acid
• CABS 4-(cyclohexylamino)-l -butanesulfonic acid
• the detergents can be denaturing detergents or non-denaturing detergents.
• denaturing detergents and non-denaturing detergents include anionic surfactants, cationic surfactants, non-ionic surfactants, zwitterionic surfactants, ampholytic surfactants, benzethonium chloride, cetyltrimethylammonium bromide (CTAB),3-[(3-cholamidopropyl)dimethylammonio]-l - propanesulfonate (CHAPS), 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-l- propanesulfonate (CHAPSO), N,N-dimethyl-decylamine-N-oxide, guanidinium thiocyanate, hexadecyltrimethylammonium bromide, lithium dodecyl sulfate (LDS), sodium dodecyl sulfate (SDS), sodium lau
• chaotropes include without limitation formamide, guanidine and salts thereof (e.g., guanidinium hydrochloride), isothiocyanate, urea, and analogs, derivatives and salts thereof.
• the composition containing the biological material comprises an oxygen radical scavenger.
• the concentration of the oxygen radical scavenger by mass (mg) relative to the volume ( ⁇ .) of the at least one alcohol solvent (or the at least one ionic organic solvent in other embodiments of a composition comprising a biological material in a substantially water- free fluid medium, as described below) is at least about 1%, 5%, 10%, 25%, 50%, 75%, 100%, 125%, 150%, 200%, 250% or 300%.
• the concentration of the oxygen radical scavenger by mass (mg) relative to the volume ( ⁇ .) of the at least one alcohol solvent (or the at least one ionic organic solvent in other embodiments) is about 25%-150%, 25%-125%, 25%- 100%, 25%-75% or 25%-50%.
• the composition comprises the biological material and an oxygen radical scavenger in ethylene glycol, 1,3 -propanediol, glycerol or 1 ,2-butanediol, or any combination thereof.
• the oxygen radical scavenger is mannitol, mannose, sucrose or trehalose or sorbitol.
• the composition containing the biological material comprises one or more substances that enhance the stability of single-stranded and double-stranded polynucleotides containing RNA nucleotides and/or DNA nucleotides.
• the composition comprises: (a) a metal chelator, a hydroxyl radical scavenger, and an RNase inhibitor; or (b) a hydroxyl radical scavenger and a DNase inhibitor.
• the composition containing the biological material comprises a metal salt, optionally in addition to one or more other substances described herein.
• the metal salt can enhance the stability and/or the solubility of the biological material in the substantially water-free fluid medium.
• the metal salt can increase the melting temperature of a double- stranded polynucleotide containing RNA nucleotides and/or DNA nucleotides.
• the metal salt can increase the solubility and the refolding yield, and can promote retention of the activity, of a protein (e.g., an enzyme) preserved in the substantially water-free fluid medium.
• the metal salt comprises an M +1 (or monovalent) salt or an M +2 (or divalent) salt, or both.
• M +1 (or monovalent) salts include without limitation lithium salts, sodium salts and potassium salts of fluoride, chloride, bromide, iodide, acetate, formate, nitrate, perchlorate (ClO f), phosphate, sulfate, tetrafluoroborate (BF 4 ⁇ ) and thiocyanate ( ⁇ SCN), and M +2 (or divalent) salts include magnesium salts, manganese salts and calcium salts of fluoride, chloride, bromide, iodide, acetate, formate, nitrate, perchlorate, phosphate, sulfate, tetrafluoroborate and thiocyanate.
• the metal salt comprises LiCl, NaCl, KC1, MgCl 2 or MnCl 2 , or
• the biological material is soluble in the substantially water- free alcohol solvent, and thus may not need to be re-dissolved for use in fluid-phase reactions or assays, including nucleic acid amplification reactions based on PCR and analytical and diagnostic assays, such as immunoassays.
• At least about 50%, 60%, 70%, 80%, 90%, 95% or 99% of the biological material by mass is dissolved in the substantially water-free alcohol solvent, e.g., after storage of the composition comprising the biological material in a closed container (e.g., a capped tube, vial or well) at a temperature from ambient temperature to about 40 °C for at least about 1 day, 3 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, 1 year, 1.5 years or 2 years.
• a closed container e.g., a capped tube, vial or well
• At least about 80% or 90% of the biological material by mass is dissolved in the substantially water- free alcohol solvent after storage of the composition comprising the biological material in a closed container (e.g., a capped tube, vial or well) at ambient temperature for at least about 3 months or 6 months.
• a closed container e.g., a capped tube, vial or well
• the biological material is stable (e.g., retains its structural integrity) in the substantially water- free alcohol solvent at ambient temperature or higher, and thus does not need to be refrigerated or frozen during shipping or storage.
• the biological material is stable (e.g., retains its structural integrity) in the substantially water-free alcohol solvent after storage of the composition comprising the biological material in a closed container (e.g., a capped tube, vial or well) at a temperature from ambient temperature to about 40 °C for at least about 1 day, 3 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, 1 year, 1.5 years or 2 years.
• the biological material is resistant to irreversible hydrolytic damage, irreversible oxidative damage, and irreversible denaturation (e.g., irreversible unfolding or irreversible loss of secondary structure or tertiary structure) after storage of the composition comprising the biological material in a closed container (e.g., a capped tube, vial or well) at a temperature from ambient temperature to about 40 °C for at least about 1 day, 3 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, 1 year, 1.5 years or 2 years.
• a closed container e.g., a capped tube, vial or well
• the biological material is resistant to irreversible hydrolytic damage, irreversible oxidative damage, and irreversible denaturation (e.g., irreversible unfolding or irreversible loss of secondary structure or tertiary structure) after storage of the composition comprising the biological material in a closed container (e.g., a capped tube, vial or well) at ambient temperature for at least about 3 months or 6 months.
• a closed container e.g., a capped tube, vial or well
• the biological material retains its function or activity when it is preserved in the substantially water-free alcohol solvent at ambient temperature or higher and is tested for its function or activity under appropriate conditions (e.g., in an aqueous medium).
• the biological material retains its function or activity after storage of the composition comprising the biological material in a closed container (e.g., a capped tube, vial or well) at a temperature from ambient temperature to about 40 °C for at least about 1 day, 3 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, 1 year, 1.5 years or 2 years.
• a closed container e.g., a capped tube, vial or well
• the biological material retains at least about 50%, 60%, 70%, 80%, 90%, 95% or 99% of its function or activity after storage of the composition comprising the biological material in a closed container (e.g., a capped tube, vial or well) at a temperature from ambient temperature to about 40 °C for at least about 1 day, 3 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, 1 year, 1.5 years or 2 years.
• the biological material retains at least about 90% of its function or activity after storage of the composition comprising the biological material in a closed container (e.g., a capped tube, vial or well) at ambient temperature for at least about 3 months or 6 months.
• the function or activity of a biological material preserved in the substantially water- free alcohol solvent and tested at a particular time point can be compared to the function or activity of a positive control, e.g., the function or activity of the biological material prepared under appropriate conditions (e.g., in an aqueous medium) shortly before its use in the test protocol (e.g., test reaction or test assay).
• a positive control e.g., the function or activity of the biological material prepared under appropriate conditions (e.g., in an aqueous medium) shortly before its use in the test protocol (e.g., test reaction or test assay).
• Non-limiting examples of retention of a biological material's function or activity include: a) a polypeptide enzyme or a polynucleotide enzyme retaining its enzymatic or catalytic function or activity; b) a polypeptide retaining its ability to regulate (e.g., agonize or antagonize/inhibit) an enzyme; c) an antibody, a polypeptide aptamer or a polynucleotide aptamer retaining its binding affinity or specificity for a target antigen, ligand or analyte; d) a polypeptide ligand of an antibody retaining its ability to be recognized and bound by the antibody; e) a hormone or a cytokine retaining its biological function or activity; f) a polypeptide therapeutic retaining its pharmacological function or activity; g) a vaccine retaining its prophylactic or immune function or activity; h) a pair of forward and reverse primers retaining their ability to prime amplification of
• Further embodiments of the disclosure relate to a method of preserving a biological material in a substantially water-free, non-ionic organic solvent (e.g., an alcohol solvent).
• a substantially water-free, non-ionic organic solvent e.g., an alcohol solvent.
• the method comprises: a) mixing an aqueous mixture comprising a polypeptide, a polynucleotide or a biological sample, or any combination thereof, with at least one alcohol solvent to produce an aqueous organic mixture, wherein: the polypeptide comprises an enzyme that mediates a nucleic acid reaction, a polypeptide that regulates an enzyme, an antibody, a polypeptide ligand of an antibody, a polypeptide aptamer, a protein or enzyme useful for detection, a toxin, a hormone, a cytokine, a polypeptide therapeutic or a vaccine, or a derivative thereof or any combination thereof; the polynucleotide comprises a polynucleotide used in a nucleic acid reaction, a catalytic
• the biological sample comprises a biological fluid, a biological suspension, a fluid aspirate, blood, plasma, serum, lymph, cerebrospinal fluid, gastric fluid, bile, perspiration, ocular fluid, tears, oral fluid, sputum, saliva, a buccal sample, a tonsil sample, a nasal sample, mucus, a nasopharyngeal sample, semen, urine, a vaginal sample, a cervical sample, a rectal sample, a fecal sample, a wound or purulent sample, hair, a tissue, a tissue homogenate, cells, a cellular lysate, a tissue or cell biopsy, skin cells, tumor or cancer cells, a microbe, a pathogen, a bacterium, a fungus, a protozoan or a virus, or any combination thereof
• the at least one alcohol solvent in the composition comprises no more than about 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% or 0.5% water by mass relative to the combined mass of water and the at least one alcohol solvent after removal of water from the aqueous organic mixture, and optionally after storage of the composition in a closed container (e.g., a capped tube, vial or well) at a temperature from ambient temperature to about 40 °C for at least about 1 day, 3 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, 1 year, 1.5 years or 2 years.
• a closed container e.g., a capped tube, vial or well
• the at least one alcohol solvent in the composition comprises no more than about 10%), 5%> or 1%> water by mass relative to the combined mass of water and the at least one alcohol solvent after removal of water from the aqueous organic mixture, and optionally after storage of the composition in a closed container (e.g., a capped tube, vial or well) at a temperature from ambient temperature to about 40 °C for at least about 1 day, 3 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, 1 year, 1.5 years or 2 years.
• a closed container e.g., a capped tube, vial or well
• the at least one alcohol solvent in the composition comprises no more than about 10%, 5% or 1% water by mass relative to the combined mass of water and the at least one alcohol solvent after removal of water from the aqueous organic mixture and after storage of the composition in a closed container (e.g., a capped tube, vial or well) at ambient temperature for at least about 3 months or 6 months.
• a closed container e.g., a capped tube, vial or well
• the at least one alcohol solvent can comprise one or more of any of the alcohol solvents described herein.
• the polypeptide, the polynucleotide or the biological sample, or any combination thereof can comprise any polypeptide, any polynucleotide or any biological sample described herein.
• water is removed from the aqueous organic mixture by evaporation. In further embodiments, removing water from the aqueous organic mixture to produce the composition in a fluid state does not pass through an intermediate solid state. In some embodiments, water is removed from the aqueous organic mixture at ambient temperature or at reduced temperature but above the freezing point of the aqueous organic mixture or the at least one alcohol solvent. In further embodiments, water is removed from the aqueous organic mixture at ambient pressure (e.g., at about 1 atm) or at reduced pressure (e.g., at about 0.1 atm, 0.2 atm, 0.3 atm, 0.4 atm, or 0.5 atm or greater).
• ambient pressure e.g., at about 1 atm
• reduced pressure e.g., at about 0.1 atm, 0.2 atm, 0.3 atm, 0.4 atm, or 0.5 atm or greater.
• water is removed from the aqueous organic mixture at ambient temperature and ambient pressure. In other embodiments, water is removed from the aqueous organic mixture at ambient temperature and reduced pressure. In yet other embodiments, water is removed from the aqueous organic mixture at reduced pressure and at reduced temperature but above the freezing point of the aqueous organic mixture or the at least one alcohol solvent. In further embodiments, water is removed from the aqueous organic mixture at a relative humidity of no more than about 60%, 50%, 40%, 30% or 20%.
• the composition can be any composition comprising a biological material in a substantially water- free alcohol solvent as described herein.
• the composition can comprise one or more substances as described herein, where the aqueous mixture, the at least one alcohol solvent or the aqueous organic mixture, or any combination thereof, can comprise the one or more substances (e.g., the one or more substances can be added to the aqueous mixture, the at least one alcohol solvent or the aqueous organic mixture, or any combination thereof).
• the one or more substances comprise: (a) a protease inhibitor; (b) an oxygen radical scavenger; (c) a metal chelator, a hydroxyl radical scavenger, and an RNase inhibitor; or (d) a hydroxyl radical scavenger and a DNase inhibitor.
• the composition can comprise a metal salt as described herein, where the aqueous mixture, the at least one alcohol solvent or the aqueous organic mixture, or any combination thereof, can comprise the metal salt (e.g., the metal salt can be added to the aqueous mixture, the at least one alcohol solvent or the aqueous organic mixture, or any combination thereof).
• composition can be re-hydrated by addition of an aqueous solution (e.g., water or an aqueous buffer) shortly before the composition is to be used in a biochemical reaction (e.g., PCR) or an analysis (e.g., an immunoassay).
• aqueous solution e.g., water or an aqueous buffer
• an analysis e.g., an immunoassay
• compositions comprising a biological material in an anhydrous, ionic organic solvent
• compositions comprising a biological material in a substantially water- free, ionic organic solvent.
• a composition comprises: a) a polypeptide, a polynucleotide or a biological sample, or any combination thereof, wherein the polypeptide comprises an enzyme that mediates a nucleic acid reaction, a polypeptide that regulates an enzyme, an antibody, a polypeptide ligand of an antibody, a polypeptide aptamer, a protein or enzyme useful for detection, a toxin, a hormone, a cytokine, a polypeptide therapeutic or a vaccine, or a derivative thereof or any combination thereof; wherein the polypeptide comprises an enzyme that mediates a nucleic acid reaction, a polypeptide that regulates an enzyme, an antibody, a polypeptide ligand of an antibody, a polypeptide aptamer, a protein or enzyme useful for detection, a toxin, a hormone, a cytokine, a polypeptide therapeutic or a vaccine,
• polynucleotide comprises a polynucleotide used in a nucleic acid reaction, a catalytic polynucleotide, or a polynucleotide that binds specifically to a target ligand, or a derivative thereof or any combination thereof; and wherein the biological sample comprises a biological fluid, a biological suspension, a fluid aspirate, blood, plasma, serum, lymph, cerebrospinal fluid, gastric fluid, bile, perspiration, ocular fluid, tears, oral fluid, sputum, saliva, a buccal sample, a tonsil sample, a nasal sample, mucus, a nasopharyngeal sample, semen, urine, a vaginal sample, a cervical sample, a rectal sample, a fecal sample, a wound or purulent sample, hair, a tissue, a tissue homogenate, cells, a cellular lysate, a tissue or cell biopsy, skin cells, tumor or cancer cells,
• the at least one ionic organic solvent in the composition comprises no more than about 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% or 0.5% water by mass relative to the combined mass of water and the at least one ionic organic solvent, e.g., after storage of the composition in a closed container (e.g., a capped tube, vial or well) at a temperature from ambient temperature to about 40 °C for at least about 1 day, 3 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, 1 year, 1.5 years or 2 years.
• a closed container e.g., a capped tube, vial or well
• the at least one ionic organic solvent in the composition comprises no more than about 10%>, 5% or 1%> water by mass relative to the combined mass of water and the at least one ionic organic solvent, e.g., after storage of the composition in a closed container (e.g., a capped tube, vial or well) at a temperature from ambient temperature to about 40 °C for at least about 1 day, 3 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, 1 year, 1.5 years or 2 years.
• a closed container e.g., a capped tube, vial or well
• the at least one ionic organic solvent in the composition comprises no more than about 10%>, 5% or 1% water by mass relative to the combined mass of water and the at least one ionic organic solvent after storage of the composition in a closed container (e.g., a capped tube, vial or well) at ambient temperature for at least about 3 months or 6 months.
• a closed container e.g., a capped tube, vial or well
• the at least one ionic organic solvent is substantially soluble in water - e.g., at least about 50%, 60%, 70%, 80%, 90%, 95% or 99% of the at least one ionic organic solvent by mass or volume is soluble in water. In certain embodiments, at least about 90%>, 95% or 99% of the at least one ionic organic solvent by mass or volume is soluble in water. In an
• the at least one ionic organic solvent is miscible with water. Solubility of the at least one ionic organic solvent in water promotes transfer of a biological material from an aqueous medium to the at least one ionic organic solvent.
• the at least one ionic organic solvent has a boiling point substantially greater than that of water - e.g., a boiling point at least or greater than about 125 °C, 150 °C, 175 °C, 200 °C, 250 °C or 300 °C at a pressure of about 1 atm. In certain embodiments, the at least one ionic organic solvent has a boiling point at least or greater than about 150 °C, 200 °C or 250 °C at a pressure of about 1 atm.
• the at least one ionic organic solvent having a boiling point greater than the boiling point of water allows for an aqueous mixture comprising a biological material to be mixed with at least one ionic organic solvent and for water to be selectively removed (e.g., by evaporation) from the resulting aqueous organic mixture without substantial loss of the at least one ionic organic solvent.
• the at least one ionic organic solvent has a dynamic (or absolute) viscosity of no more than about 2000, 1500, 1000, 500, 400, 300, 200, 100, 50 or 25 centipoise (cP) or mPa- s at ambient temperature. In certain embodiments, the at least one ionic organic solvent has a dynamic (or absolute) viscosity of no more than about 1000, 500, 200, 100 or 50 cP or mPa- s at ambient temperature.
• a lower dynamic (or absolute) viscosity of the at least one ionic organic solvent allows for more facile handling of the composition comprising the biological material and the at least one ionic organic solvent (e.g., using a pipette or other means of transferring the composition).
• the at least one ionic organic solvent is a eutectic solvent. In further embodiments, the at least one ionic organic solvent is a deep eutectic solvent.
• the molar ratio of the organic salt to the organic hydrogen bond donor in the at least one ionic organic solvent is from about 5: 1 to about 1 :5, or from about 4: 1 to about 1 :4, or from about 3: 1 to about 1 :3, or from about 2:1 to about 1 :2, or from about 1.5:1 to about 1 : 1.5. In certain embodiments, the molar ratio of the organic salt to the organic hydrogen bond donor in the at least one ionic organic solvent is from about 1 : 1 to about 1 :2, or is about 1 : 1, about 1 : 1.5 or about 1 :2.
• the organic salt of the at least one ionic organic solvent can be any organic salt capable of forming a solvent with an organic hydrogen bond donor (e.g., by heating a mixture of the organic salt and the organic hydrogen bond donor).
• the organic salt of the at least one ionic organic solvent comprises one or more organic salts selected from the group consisting of primary ammonium salts, secondary ammonium salts, tertiary ammonium salts, and quaternary ammonium salts.
• Examples of primary ammonium salts include without limitation methylammonium salts, ethylammonium salts, propylammonium salts, butylammonium salts, 2- hydroxyethylammonium salts, 2-acetylethylammonium salts, 2-chloroethylammonium salts, 2- fluoroethylammonium salts, and benzylammonium salts.
• Non-limiting examples of secondary ammonium salts include dimethylammonium salts, diethylammonium salts, dipropylammonium salts, dibutylammonium salts, bis(2-hydroxyethyl)- ammonium salts, dibenzylammonium salts, ethylmethylammonium salts, (2-hydroxyethyl)methyl- ammonium salts, (2-hydroxyethyl)ethylammonium salts, (2-acetylethyl)methylammonium salts, (2- acetylethyl)ethylammonium salts, (2-chloroethyl)methylammonium salts, (2-chloroethyl)ethyl- ammonium salts, (2-fluoroethyl)methylammonium salts, (2-fluoroethyl)ethylammonium salts, benzylmethylammonium salts, benzyleth
• Non-limiting examples of tertiary ammonium salts include trimethylammonium salts, triethylammonium salts, dimethylethylammonium salts, diethylmethylammonium salts,
• quaternary ammonium salts include without limitation
• the anion of the organic salt can be any anion capable of interacting (e.g., complexing or hydrogen bonding) with an organic hydrogen bond donor.
• the anion of the organic salt is a monovalent anion.
• the anion of the organic salt e.g., the primary ammonium salts, the secondary ammonium salts, the tertiary ammonium salts, and the quaternary ammonium salts described herein
• the organic salt comprises a quaternary ammonium salt.
• the organic salt comprises a (2-hydroxyethyl)trimethylammonium (choline) salt.
• the organic salt comprises choline chloride or choline acetate.
• the organic hydrogen bond donor of the at least one ionic organic solvent can be any organic hydrogen bond donor capable of forming a solvent with an organic salt, or any organic hydrogen bond donor capable of interacting (e.g., complexing or hydrogen bonding) with the anion of an organic salt.
• the organic hydrogen bond donor of the at least one ionic organic solvent comprises one or more organic hydrogen bond donors selected from the group consisting of urea compounds, thiourea compounds, carbamates, amides, carboxylic acids, phenolic compounds, acyclic alcohols, and cyclic alcohols.
• Non-limiting examples of urea compounds and thiourea compounds include urea, N- methylurea, ⁇ , ⁇ '-dimethylurea, N,N-dimethylurea, ⁇ , ⁇ , ⁇ '-trimethylurea, thiourea, N- methylthiourea, N,N'-dimethylthiourea, ⁇ , ⁇ -dimethylthiourea, and ⁇ , ⁇ , ⁇ '-trimethylthiourea.
• the organic hydrogen bond donor comprises urea.
• Examples of carbamates include without limitation methyl carbamate, ethyl carbamate, propyl carbamate, butyl carbamate, methyl N-methylcarbamate, ethyl N-methylcarbamate, propyl N- methylcarbamate, and butyl N-methylcarbamate.
• Examples of amides include without limitation acetamide, propanamide, butanamide, pentanamide, benzamide, N-methylacetamide, N- methylpropanamide, N-methylbutanamide, N-methylpentanamide, and N-methylbenzamide.
• the organic hydrogen bond donor comprises acetamide.
• Non-limiting examples of carboxylic acids include adipic acid, benzoic acid, citric acid, ethylenediaminetetraacetic acid, fumaric acid, maleic acid, malonic acid, oxalic acid, phenylacetic acid, phenylpropionic acid, propane- 1,2,3 -tricarboxylic acid (tricarballylic acid), succinic acid, and tartaric acid.
• the organic hydrogen bond donor comprises citric acid, malonic acid or oxalic acid, or any combination thereof.
• phenolic compounds include without limitation phenol and tyrosine.
• Examples of acyclic alcohols include without limitation the linear and branched C2-C6 acyclic alcohols having one or more hydroxyl groups and optionally comprising one or more halide atoms described herein.
• Examples of cyclic alcohols include without limitation ascorbic acid and the C3-C6 cyclic alcohols having one or more hydroxyl groups and three to six ring carbon atoms and optionally comprising one or more halide atoms described herein.
• the organic hydrogen bond donor comprises ethylene glycol, 1 ,2-propanediol, 1,3-propanediol, glycerol, 1 ,2-butanediol, 1,3-butanediol, 1 ,4-butanediol, 2,3-butanediol, 1 ,2,4-butanetriol or 1,5-pentanediol, or any combination thereof.
• the organic hydrogen bond donor comprises ethylene glycol, 1,3-propanediol, glycerol or 1,2-butanediol, or any combination thereof.
• ionic organic solvents include without limitation: (1) choline chloride or ethylammonium chloride and urea; (2) choline chloride or ethylammonium chloride and acetamide; (3) choline chloride or ethylammonium chloride and citric acid; (4) choline chloride or
• ethylammonium chloride and malonic acid (5) choline chloride or ethylammonium chloride and oxalic acid; (6) choline chloride or ethylammonium chloride and ethylene glycol; (7) choline chloride or ethylammonium chloride and glycerol; and (8) choline chloride or ethylammonium chloride and 1 ,2-butanediol.
• the at least one ionic organic solvent can be prepared by any method known in the art.
• the at least one ionic organic solvent can be prepared by heating at elevated temperature (e.g., at about 50 °C, 75 °C or 100 °C or higher) a mixture comprising one or more organic salts and one or more organic hydrogen bond donors with stirring to produce a liquid (e.g., a homogeneous liquid).
• elevated temperature e.g., at about 50 °C, 75 °C or 100 °C or higher
• An organic salt having a melting point above ambient temperature, and/or an organic hydrogen bond donor having a melting point above ambient temperature can be used to prepare the at least one ionic organic solvent.
• the composition can comprise in the substantially water-free ionic organic solvent any polypeptide, any polynucleotide or any biological sample, or any combination thereof, described herein.
• the composition can further comprise a metal salt, and/or one or more substances selected from the group consisting of reducing agents, antioxidants, free radical scavengers, oxygen radical scavengers, hydroxyl radical scavengers, singlet oxygen quenchers, hydroperoxide-removing agents, protease inhibitors, nuclease inhibitors, ribonuclease (RNase) inhibitors, deoxyribonuclease (DNase) inhibitors, metal chelators, preservatives, antimicrobials, buffers (or buffering agents), detergents, and chaotropes, as described herein.
• the composition comprises the biological material and: (a)
• an oxygen radical scavenger (b) an oxygen radical scavenger; (c) a metal chelator, a hydroxyl radical scavenger, and an RNase inhibitor; or (d) a hydroxyl radical scavenger and a DNase inhibitor.
• the biological material is soluble in the substantially water-free ionic organic solvent, and thus may not need to be re-dissolved for use in fluid-phase reactions or assays, including nucleic acid amplification reactions based on PCR and analytical and diagnostic assays, such as
• the biological material by mass is dissolved in the substantially water-free ionic organic solvent, e.g., after storage of the composition comprising the biological material in a closed container (e.g., a capped tube, vial or well) at a temperature from ambient temperature to about 40 °C for at least about
• a closed container e.g., a capped tube, vial or well
• At least about 80%> or 90%> of the biological material by mass is dissolved in the substantially water-free ionic organic solvent after storage of the composition comprising the biological material in a closed container (e.g., a capped tube, vial or well) at ambient temperature for at least about 3 months or 6 months.
• a closed container e.g., a capped tube, vial or well
• the biological material is stable (e.g., retains its structural integrity) in the substantially water- free ionic organic solvent at ambient temperature or higher, and thus does not need to be refrigerated or frozen during shipping or storage.
• the biological material is stable (e.g., retains its structural integrity) in the substantially water-free ionic organic solvent after storage of the composition comprising the biological material in a closed container (e.g., a capped tube, vial or well) at a temperature from ambient temperature to about 40 °C for at least about 1 day, 3 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, 1 year, 1.5 years or 2 years.
• the biological material is resistant to irreversible hydrolytic damage, irreversible oxidative damage, and irreversible denaturation (e.g., irreversible unfolding or irreversible loss of secondary structure or tertiary structure) after storage of the composition comprising the biological material in a closed container (e.g., a capped tube, vial or well) at a temperature from ambient temperature to about 40 °C for at least about 1 day, 3 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, 1 year, 1.5 years or 2 years.
• a closed container e.g., a capped tube, vial or well
• the biological material is resistant to irreversible hydrolytic damage, irreversible oxidative damage, and irreversible denaturation (e.g., irreversible unfolding or irreversible loss of secondary structure or tertiary structure) after storage of the composition comprising the biological material in a closed container (e.g., a capped tube, vial or well) at ambient temperature for at least about 3 months or 6 months.
• a closed container e.g., a capped tube, vial or well
• the biological material retains its function or activity when it is preserved in the substantially water-free ionic organic solvent at ambient temperature or higher and is tested for its function or activity under appropriate conditions (e.g., in an aqueous medium).
• the biological material retains its function or activity after storage of the composition comprising the biological material in a closed container (e.g., a capped tube, vial or well) at a temperature from ambient temperature to about 40 °C for at least about 1 day, 3 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, 1 year, 1.5 years or 2 years.
• a closed container e.g., a capped tube, vial or well
• the biological material retains at least about 50%, 60%, 70%, 80%, 90%, 95% or 99% of its function or activity after storage of the composition comprising the biological material in a closed container (e.g., a capped tube, vial or well) at a temperature from ambient temperature to about 40 °C for at least about 1 day, 3 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, 1 year, 1.5 years or 2 years.
• the biological material retains at least about 90% of its function or activity after storage of the composition comprising the biological material in a closed container (e.g., a capped tube, vial or well) at ambient temperature for at least about 3 months or 6 months.
• the function or activity of a biological material preserved in the substantially water- free ionic organic solvent and tested at a particular time point can be compared to the function or activity of a positive control, e.g., the function or activity of the biological material prepared under appropriate conditions (e.g., in an aqueous medium) shortly before its use in the test protocol (e.g., test reaction or test assay).
• a positive control e.g., the function or activity of the biological material prepared under appropriate conditions (e.g., in an aqueous medium) shortly before its use in the test protocol (e.g., test reaction or test assay).
• Additional embodiments of the disclosure relate to a method of preserving a biological material in a substantially water- free, ionic organic solvent.
• the method comprises: a) mixing an aqueous mixture comprising a polypeptide, a polynucleotide or a biological sample, or any combination thereof, with at least one ionic organic solvent to produce an aqueous organic mixture, wherein: the polypeptide comprises an enzyme that mediates a nucleic acid reaction, a polypeptide that regulates an enzyme, an antibody, a polypeptide ligand of an antibody, a polypeptide aptamer, a protein or enzyme useful for detection, a toxin, a hormone, a cytokine, a polypeptide therapeutic or a vaccine, or a derivative thereof or any combination thereof;
• the polynucleotide comprises a polynucleotide used in a nucleic acid reaction, a catalytic
• the biological sample comprises a biological fluid, a biological suspension, a fluid aspirate, blood, plasma, serum, lymph, cerebrospinal fluid, gastric fluid, bile, perspiration, ocular fluid, tears, oral fluid, sputum, saliva, a buccal sample, a tonsil sample, a nasal sample, mucus, a nasopharyngeal sample, semen, urine, a vaginal sample, a cervical sample, a rectal sample, a fecal sample, a wound or purulent sample, hair, a tissue, a tissue homogenate, cells, a cellular lysate, a tissue or cell biopsy, skin cells, tumor or cancer cells, a microbe, a pathogen, a bacterium, a fungus, a protozoan or a virus, or any combination thereof
• the at least one ionic organic solvent in the composition comprises no more than about 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% or 0.5% water by mass relative to the combined mass of water and the at least one ionic organic solvent after removal of water from the aqueous organic mixture, and optionally after storage of the composition in a closed container (e.g., a capped tube, vial or well) at a temperature from ambient temperature to about 40 °C for at least about
• a closed container e.g., a capped tube, vial or well
• the at least one ionic organic solvent in the composition comprises no more than about 10%>, 5% or 1%> water by mass relative to the combined mass of water and the at least one ionic organic solvent after removal of water from the aqueous organic mixture, and optionally after storage of the composition in a closed container (e.g., a capped tube, vial or well) at a temperature from ambient temperature to about 40 °C for at least about 1 day, 3 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, 1 year, 1.5 years or 2 years.
• a closed container e.g., a capped tube, vial or well
• the at least one ionic organic solvent in the composition comprises no more than about 10%), 5%> or 1%) water by mass relative to the combined mass of water and the at least one ionic organic solvent after removal of water from the aqueous organic mixture and after storage of the composition in a closed container (e.g., a capped tube, vial or well) at ambient temperature for at least about 3 months or 6 months.
• a closed container e.g., a capped tube, vial or well
• the at least one ionic organic solvent can comprise any ionic organic solvent described herein.
• the polypeptide, the polynucleotide or the biological sample, or any combination thereof can comprise any polypeptide, any polynucleotide or any biological sample described herein.
• water is removed from the aqueous organic mixture by evaporation. In further embodiments, removing water from the aqueous organic mixture to produce the composition in a fluid state does not pass through an intermediate solid state. In some embodiments, water is removed from the aqueous organic mixture at ambient temperature or at reduced temperature but above the freezing point of the aqueous organic mixture or the at least one ionic organic solvent. In further embodiments, water is removed from the aqueous organic mixture at ambient pressure (e.g., at about 1 atm) or at reduced pressure (e.g., at about 0.1 atm, 0.2 atm, 0.3 atm, 0.4 atm, or 0.5 atm or greater).
• ambient pressure e.g., at about 1 atm
• reduced pressure e.g., at about 0.1 atm, 0.2 atm, 0.3 atm, 0.4 atm, or 0.5 atm or greater.
• water is removed from the aqueous organic mixture at ambient temperature and ambient pressure. In other embodiments, water is removed from the aqueous organic mixture at ambient temperature and reduced pressure. In yet other embodiments, water is removed from the aqueous organic mixture at reduced pressure and at reduced temperature but above the freezing point of the aqueous organic mixture or the at least one ionic organic solvent. In further embodiments, water is removed from the aqueous organic mixture at a relative humidity of no more than about 60%, 50%, 40%, 30% or 20%.
• the composition can be any composition comprising a biological material in a substantially water- free ionic organic solvent as described herein.
• the composition can comprise one or more substances as described herein, where the aqueous mixture, the at least one ionic organic solvent or the aqueous organic mixture, or any combination thereof, can comprise the one or more substances (e.g., the one or more substances can be added to the aqueous mixture, the at least one ionic organic solvent or the aqueous organic mixture, or any combination thereof).
• the one or more substances comprise: (a) a protease inhibitor; (b) an oxygen radical scavenger; (c) a metal chelator, a hydroxyl radical scavenger, and an RNase inhibitor; or (d) a hydroxyl radical scavenger and a DNase inhibitor.
• the composition can comprise a metal salt as described herein, where the aqueous mixture, the at least one ionic organic solvent or the aqueous organic mixture, or any combination thereof, can comprise the metal salt (e.g., the metal salt can be added to the aqueous mixture, the at least one ionic organic solvent or the aqueous organic mixture, or any combination thereof).
• composition can be re-hydrated by addition of an aqueous solution (e.g., water or an aqueous buffer) shortly before the composition is to be used in a biochemical reaction (e.g., PCR) or an analysis (e.g., an immunoassay).
• aqueous solution e.g., water or an aqueous buffer
• an analysis e.g., an immunoassay
• Containers and kits comprising biological materials in anhydrous fluid media
• a container can comprise any composition comprising a biological material in a substantially water-free fluid medium described herein.
• the container can be any vessel suitable for holding or storing a fluid composition.
• the container is a tube, a vial, a well or a chamber (including a well or chamber in a cartridge).
• the container is any vessel suitable for keeping away moisture during storage of a composition, such as a capped tube, vial, well or chamber.
• a capped container can have any suitable cap, such as a snap-on cap or a screw cap.
• the container is a screw-cap tube or a screw-cap vial.
• a screw-cap tube or a screw-cap vial can have a gasket for improved sealing of the screw cap to the tube or the vial.
• a kit can contain one or more compositions comprising a biological material in a substantially water- free fluid medium described herein.
• the kit can contain one or more containers comprising one or more compositions, as described herein.
• a kit can contain reagents for performing a biochemical reaction (e.g., a nucleic acid amplification reaction, such as PCR or RT- PCR) or an assay (e.g., an immunoassay, such as an ELISA or a sandwich immunoassay), wherein: 1) the kit can include a container containing a composition comprising all the reagents for performing the biochemical reaction or the assay; or
• the kit can include two or more containers, where each container contains a composition comprising one or more reagents for performing the biochemical reaction or the assay as described herein, and the one or more reagents in each of the containers can be combined prior to or at the time of their use in the biochemical reaction or the assay or can be used at different times in performing the biochemical reaction or the assay as appropriate.
• kits can further comprise a substrate with which the enzyme can react to produce a detectable signal (e.g., a color change in the substrate).
• a detectable signal e.g., a color change in the substrate
• a kit can comprise reagents for performing PCR or RT-PCR, wherein: 1) the kit can include a container containing a composition comprising all the reagents for performing PCR or RT-PCR; or 2) the kit can include two or more containers, where each container contains a composition comprising one or more reagents for performing PCR or RT-PCR as described herein (e.g., a DNA polymerase in one container and at least one pair of forward and reverse primers in a separate container for PCR, or a reverse transcriptase and a DNA polymerase in one container and at least one reverse transcription primer and at least one pair of forward and reverse primers in a separate container for RT-PCR), and the one or more reagents in each of the containers can be combined prior to or at the time of their use in PCR or RT-PCR.
• a DNA polymerase in one container and at least one pair of forward and reverse primers in a separate container for PCR or a reverse transcriptase and a DNA polyme
• kits can comprise reagents for performing an ELISA or a sandwich immunoassay, wherein: 1) the kit can include two or more containers, each container containing a composition comprising one or more reagents for performing the ELISA or the sandwich
• the immunoassay as described herein e.g., a first antibody in one container and a second antibody in a separate container for an ELISA or a sandwich immunoassay
• the one or more reagents in each of the containers can be used at different times in performing the ELISA or the sandwich immunoassay as appropriate (e.g., the second antibody can be used subsequent to use of the first antibody in the ELISA or the sandwich immunoassay).
• a kit can further comprise water or an aqueous solution (e.g., an aqueous buffer) in a container (e.g., a vial, bottle or cartridge) for re-hydration of the biological material in a composition for use, e.g., in a reaction (e.g., a PCR amplification reaction) or an assay (e.g., an analytical or diagnostic assay, such as an immunoassay) which is performed in an aqueous medium.
• a kit can comprise a desiccant for promoting preservation of the biological material in a substantially anhydrous state.
• Non-limiting examples of desiccants include activated alumina, aerogel, silica gel, benzophenone, calcium chloride, calcium sulfate, cobalt chloride, copper sulfate, lithium chloride, lithium bromide, magnesium chloride, magnesium perchlorate, magnesium sulfate, potassium carbonate, sodium chlorate, sodium chloride, sodium hydroxide, sodium sulfate, sucrose, clay (e.g., bentonite clay and montmorillonite clay), and molecular sieves.
• a kit can comprise instruction for storing and using a composition, and optionally instruction for using water or an aqueous solution (e.g., an aqueous buffer) to re -hydrate the biological material in a composition.
• the disclosure provides a liquid composition
• a liquid composition comprising: (a) one or more linear or branched C2-C6 acyclic alcohols having two or more hydroxyl groups, wherein the one or more alcohols each has a boiling point greater than 125 °C; and (b) one or more proteins having biological function; wherein the composition comprises no more than about 20% water by mass relative to the combined mass of water and the alcohols, and each protein retains its function (e.g., after rehydration).
• the one or more alcohols comprise the predominant liquid in the composition, e.g., at least 60%, 70%, 80%, 90%, 95%, 98% or 99% by mass.
• At least one of the alcohols is selected from 1 ,2-ethanediol (ethylene glycol), 1 ,2- propanediol (propylene glycol), 1,3 -propanediol, 1,2,3-propanetriol (glycerol), 1 ,2-butanediol, 1,3- butanediol, 1 ,4-butanediol, 2,3-butanediol, 1,2,3-butanetriol, 1 ,2,4-butanetriol, 1 ,2-pentanediol, 1,3- pentanediol, 1 ,4-pentanediol, 1,5-pentanediol, 2,3-pentanediol, 2,4-pentanediol, 1,2,3-pentanetriol, 1 ,2,4-pentanetriol, 1,2,5-pentanetriol, 1,3,4-pentanetriol, 1,
• the composition comprises no more than about 15%>, 10%>, 5%>, 4%>, 3%>, 2%>, 1%> or 0.5%> water by mass relative to the combined mass of water and the alcohols.
• at least one of the alcohols has a boiling point above 150 °C, 175 °C, 200 °C or 250 °C at a pressure of about 1 atmosphere (atm).
• the protein is an enzyme or a binding protein (e.g., an antibody).
• the one or more proteins are a plurality of proteins.
• a plurality of the proteins comprise a first enzyme that catalyzes reaction of a substate to a first product, and a second enzyme that catalyzes reaction of the first product to a second product.
• a plurality of proteins comprise a first and second binding protein that each bind to the same protein or some other non-protein anayte and are configured for performing a sandwich immunoassay.
• the composition comprises biomolecules sufficient to perform PCR when a substrate is added, sufficient to perform reverse transcriptase PCR when a substrate is added, or sufficient to perform adaptor ligation when a DNA substrate is added.
• the composition further comprises an oxygen radical scavenger.
• the composition further comprises an anti-oxidant.
• the disclosure provides a method comprising a) combining a liquid composition comprising one or more linear or branched C2-C6 acyclic alcohols having two or more hydroxyl groups, wherein the one or more alcohols each has a boiling point greater than 125 °C; and one or more proteins having biological function; wherein the composition comprises no more than about 20% water by mass relative to the combined mass of water and the alcohols; and b) removing water from the combination to produce a composition substantially free of water, wherein each protein retains its function (e.g., after rehydration).
• removing water comprises subjecting the combination to sub-atmospheric pressure to evaporate the water (e.g., at no more than about 0.1 atm, 0.2 atm, 0.3 atm, 0.4 atm, or 0.5 atm, or 07 atm, 0.9 atm, or 0.9 atm.
• removing water comprises subjecting the combination to a temperature at least 10°C are above room temperature.
• method further comprises storing a composition substantially free of water for at least one day, one week, one month, or one year before rehydration.
• the composition is stored at room temperature, e.g., between about 21°C and 25°C.
• the composition is stored below room temperature e.g., below 0°C.
• the method further comprises rehydrating the stored composition to restore function of the protein.
• this disclosure provides a method comprising: 1) providing a composition comprising comprising (a) one or more linear or branched C2-C6 acyclic alcohols having two or more hydroxyl groups, wherein the one or more alcohol each has a boiling point greater than 125 °C; and (b) one or more proteins; wherein the composition comprises no more than about 20% water by mass relative to the combined mass of water and the alcohols, and each protein retains its structural integrity, function and activity; 2) hydrating the composition with an amount of water sufficient to restore biological activity of one or more of the proteins; 3) contacting the at least one protein with a substrate or an analyte; and 4) performing a reaction on the substrate or binding the analyte with at least one of the proteins.
• the method comprises performing a reaction on a DNA substrate, wherein the action comprises PCR, rtPCR or adaptor ligation.
• the method comprises binding an analyte in the performance of immunoassay.
• composition comprising:
• the polypeptide comprises an enzyme that mediates a nucleic acid reaction, a polypeptide that regulates an enzyme, an antibody, a polypeptide ligand of an antibody, a polypeptide aptamer, a protein or enzyme useful for detection, a toxin, a hormone, a cytokine, a polypeptide therapeutic or a vaccine, or a derivative thereof or any combination thereof;
• the polynucleotide comprises a polynucleotide used in a nucleic acid reaction, a catalytic polynucleotide, or a polynucleotide that binds specifically to a target ligand, or a derivative thereof or any combination thereof;
• the biological sample comprises a biological fluid, a biological suspension, a fluid aspirate, blood, plasma, serum, lymph, cerebrospinal fluid, gastric fluid, bile, perspiration, ocular fluid, tears, oral fluid, sputum, saliva, a buccal sample, a tonsil sample, a nasal sample, mucus, a nasopharyngeal sample, semen, urine, a vaginal sample, a cervical sample, a rectal sample, a fecal sample, a wound or purulent sample, hair, a tissue, a tissue homogenate, cells, a cellular lysate, a tissue or cell biopsy, skin cells, tumor or cancer cells, a microbe, a pathogen, a bacterium, a fungus, a protozoan or a virus, or any combination thereof; and
• At least one alcohol solvent selected from the group consisting of linear and branched C2- Ce acyclic alcohols having one or more hydroxyl groups and C3-C6 cyclic alcohols having one or more hydroxyl groups and three to six ring carbon atoms, wherein the acyclic alcohols and the cyclic alcohols optionally comprise one or more halide atoms;
• composition is in a fluid state and is substantially free of water.
• composition of embodiment 1 wherein the at least one alcohol solvent in the
• composition comprises no more than about 20%, 10%, 5% or 2% water by mass relative to the combined mass of water and the at least one alcohol solvent.
• composition of any one of the preceding embodiments, wherein at least about 50%, 60%>, 70%), 80%), 90%) or 95% of the at least one alcohol solvent by volume is soluble in water.
• composition of embodiment 3, wherein the at least one alcohol solvent is miscible with water.
• cP centipoise
• composition of embodiment 7, wherein the linear and branched C2-C5 acyclic alcohols having one hydroxyl group and optionally comprising one or more halide atoms are selected from the group consisting of 2-chloroethanol, 2,2-dichloroethanol, 1 -butanol, 1 -pentanol, 2-methylbutan-l -ol, 3-methylbutan-l -ol, 2,2-dimethylpropan-l -ol, 2-pentanol, 3-methylbutan-2-ol, and 3-pentanol.
• the linear and branched C 2 -C 6 acyclic alcohols having two or three hydroxyl groups are selected from the group consisting of 1,2-ethanediol (ethylene glycol), 1 ,2-propanediol (propylene glycol), 1,3 -propanediol, 1,2,3-propanetriol (glycerol), 1 ,2-butanediol, 1,3-butanediol, 1 ,4-butanediol, 2,3-butanediol, 1,2,3-butanetriol, 1 ,2,4-butanetriol,
• composition of embodiment 10, wherein the at least one alcohol solvent comprises ethylene glycol, 1 ,2-propanediol, 1,3-propanediol, glycerol, 1 ,2-butanediol, 1,3-butanediol, 1 ,4- butanediol, 2,3-butanediol, 1 ,2,4-butanetriol or 1,5-pentanediol, or any combination thereof.
• composition of embodiment 11, wherein the at least one alcohol solvent comprises ethylene glycol, 1,3-propanediol, glycerol or 1,2-butanediol, or any combination thereof.
• composition of any one of the preceding embodiments, wherein the C 3 -C 6 cyclic alcohols having one or more hydroxyl groups are C 4 or C 5 cyclic alcohols having one or more hydroxyl groups.
• composition of embodiment 14, wherein the at least one alcohol solvent comprises cyclobutanol or cyclopentanol, or both.
• composition of any one of the preceding embodiments, wherein the at least one alcohol solvent comprises two or more alcohol solvents.
• composition of embodiment 16, wherein the at least one alcohol solvent comprises two or more alcohol solvents selected from the group consisting of ethylene glycol, 1 ,2-propanediol, 1,3- propanediol, glycerol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 2,3-butanediol, 1,2,4- butanetriol, and 1,5-pentanediol.
• composition of embodiment 17, wherein the at least one alcohol solvent comprises: a) ethylene glycol and 1,3-propanediol; or
• composition of any one of the preceding embodiments which comprises an enzyme that mediates a nucleic acid reaction.
• the enzyme that mediates a nucleic acid reaction comprises a topoisomerase, a helicase, a DNA polymerase, a reverse transcriptase, an RNA polymerase, a DNA ligase, an RNA ligase, a DNA repair enzyme, an RNA repair enzyme, an endonuclease, an exonuclease, a deoxyribonuclease (DNase), a ribonuclease (RNase), a transposase, a restriction enzyme or a nicking enzyme, or any combination thereof.
• DNase deoxyribonuclease
• RNase ribonuclease
• composition of embodiment 20, wherein the enzyme that mediates a nucleic acid reaction comprises a DNA polymerase, a reverse transcriptase or an RNA polymerase, or any combination thereof.
• composition of embodiment 21, wherein the enzyme that mediates a nucleic acid reaction comprises a DNA polymerase (e.g., a thermostable DNA polymerase, such as a Taq polymerase) used in polymerase chain reaction (PCR) or a reverse transcriptase used in PCR, or both.
• a DNA polymerase e.g., a thermostable DNA polymerase, such as a Taq polymerase
• PCR polymerase chain reaction
• reverse transcriptase used in PCR, or both.
• composition of any one of the preceding embodiments which comprises a polynucleotide used in a nucleic acid reaction.
• composition of embodiment 23, wherein the polynucleotide comprises at least one primer used in PCR or reverse transcription.
• composition of embodiment 24, wherein the at least one primer used in PCR or reverse transcription comprises at least one pair of a forward primer and a reverse primer for amplifying at least one nucleic acid (e.g., genetic) locus, or at least one reverse transcription primer for reverse transcribing at least one polyribonucleotide, or both; and
• the at least one pair of forward primer and reverse primer optionally is labeled with a dye (e.g., a fluorescent dye) or the at least one reverse transcription primer optionally is labeled with a dye (e.g., a fluorescent dye), or both.
• a dye e.g., a fluorescent dye
• the at least one reverse transcription primer optionally is labeled with a dye (e.g., a fluorescent dye), or both.
• composition of embodiment 25, wherein the at least one primer used in PCR comprises at least 5 or 10 different pairs of forward and reverse primers for amplifying at least 5 or 10 different short tandem repeat (STR) loci utilized in a forensic database (e.g., CODIS), and
• STR short tandem repeat
• each of the at least 5 or 10 different pairs of forward and reverse primers optionally is labeled with a dye (e.g., a fluorescent dye).
• a dye e.g., a fluorescent dye
• composition of embodiment 26, wherein the at least one primer used in PCR comprises 16 different pairs of forward and reverse primers for amplifying all 13 CODIS STR loci, Penta D, Penta E and amelogenin, and
• each of the 16 different pairs of forward and reverse primers optionally is labeled with a dye (e.g., a fluorescent dye).
• a dye e.g., a fluorescent dye
• composition of any one of the preceding embodiments which comprises one or more reagents for performing PCR
• the reagents for performing PCR comprise a DNA polymerase and at least one pair of a forward primer and a reverse primer for amplifying at least one nucleic acid (e.g., genetic) locus
• the at least one pair of forward primer and reverse primer optionally is labeled with a dye (e.g., a fluorescent dye).
• composition of embodiment 28, wherein the at least one pair of forward and reverse primers comprises at least 5 or 10 different pairs of forward and reverse primers for amplifying at least 5 or 10 different STR loci utilized in a forensic database (e.g., CODIS), and
• each of the at least 5 or 10 different pairs of forward and reverse primers optionally is labeled with a dye (e.g., a fluorescent dye).
• a dye e.g., a fluorescent dye
• composition of embodiment 29, wherein the at least one pair of forward and reverse primers comprises 16 different pairs of forward and reverse primers for amplifying all 13 CODIS STR loci, Penta D, Penta E and amelogenin, and
• each of the 16 different pairs of forward and reverse primers optionally is labeled with a dye (e.g., a fluorescent dye).
• a dye e.g., a fluorescent dye
• a thermostable DNA polymerase e.g., a Taq polymerase
• composition of any one of embodiments 28 to 31, wherein the reagents for performing PCR further comprise deoxyribonucleotide triphosphates and optionally a buffer and/or a metal salt (e.g., magnesium chloride).
• a buffer and/or a metal salt e.g., magnesium chloride
• composition of embodiment 32 which comprises the DNA polymerase, the at least one pair of forward and reverse primers, deoxyribonucleotide triphosphates, and optionally a buffer and/or a metal salt (e.g., magnesium chloride).
• a metal salt e.g., magnesium chloride
• composition of embodiment 32 which comprises the DNA polymerase and no primer.
• composition of embodiment 34 which further comprises deoxyribonucleotide triphosphates and optionally a buffer and/or a metal salt (e.g., magnesium chloride).
• a buffer and/or a metal salt e.g., magnesium chloride
• composition of embodiment 32 which comprises the at least one pair of forward and reverse primers and no DNA polymerase.
• composition of embodiment 36 which further comprises deoxyribonucleotide triphosphates and optionally a buffer and/or a metal salt (e.g., magnesium chloride).
• a buffer and/or a metal salt e.g., magnesium chloride
• composition of embodiment 36 or 37 which is provided in a kit comprising the composition of embodiment 34 or 35.
• the reagents for performing reverse transcription comprise a reverse transcriptase and at least one reverse transcription primer for reverse transcribing at least one polyribonucleotide;
• the at least one reverse transcription primer optionally is labeled with a dye (e.g., a fluorescent dye).
• a dye e.g., a fluorescent dye
• composition of embodiment 39, wherein the reagents for performing reverse transcription further comprise deoxynbonucleotide triphosphates and optionally a buffer and/or a metal salt (e.g., magnesium chloride).
• a metal salt e.g., magnesium chloride
• composition of embodiment 40 which comprises the reverse transcriptase, the at least one reverse transcription primer, deoxyribonucleotide triphosphates and optionally a buffer and/or a metal salt (e.g., magnesium chloride).
• a metal salt e.g., magnesium chloride
• composition of embodiment 40 which comprises the reverse transcriptase and no reverse transcription primer.
• composition of embodiment 42 which further comprises deoxyribonucleotide triphosphates and optionally a buffer and/or a metal salt (e.g., magnesium chloride).
• a buffer and/or a metal salt e.g., magnesium chloride
• composition of embodiment 40 which comprises the at least one reverse transcription primer and no reverse transcriptase.
• composition of embodiment 44 which further comprises deoxyribonucleotide triphosphates and optionally a buffer and/or a metal salt (e.g., magnesium chloride).
• a buffer and/or a metal salt e.g., magnesium chloride
• composition of embodiment 44 or 45 which is provided in a kit comprising the composition of embodiment 42 or 43.
• the reagents for performing RT-PCR comprise a reverse transcriptase, a DNA polymerase, at least one reverse transcription primer for reverse transcribing at least one polyribonucleotide to produce at least one polydeoxyribonucleotide complementary to the at least one polyribonucleotide, and at least one pair of a forward primer and a reverse primer for amplifying the at least one complementary polydeoxyribonucleotide; and
• the at least one reverse transcription primer optionally is labeled with a dye (e.g., a fluorescent dye) and the at least one pair of forward primer and reverse primer optionally is labeled with a dye (e.g., a fluorescent dye).
• a dye e.g., a fluorescent dye
• composition of embodiment 47 wherein the DNA polymerase comprises a thermostable DNA polymerase (e.g., a Tag polymerase).
• the reagents for performing RT-PCR further comprise deoxyribonucleotide triphosphates and optionally a buffer and/or a metal salt (e.g., magnesium chloride).
• composition of embodiment 49 which comprises the reverse transcriptase, the DNA polymerase, the at least one reverse transcription primer, the at least one pair of forward and reverse primers, deoxyribonucleotide triphosphates, and optionally a buffer and/or a metal salt (e.g., magnesium chloride).
• a metal salt e.g., magnesium chloride
• composition of embodiment 49 which comprises the reverse transcriptase, the DNA polymerase, no reverse transcription primer, and no pair of forward and reverse primers.
• composition of embodiment 51 which further comprises deoxyribonucleotide triphosphates and optionally a buffer and/or a metal salt (e.g., magnesium chloride).
• a buffer and/or a metal salt e.g., magnesium chloride
• composition of embodiment 49 which comprises the at least one reverse transcription primer, the at least one pair of forward and reverse primers, no reverse transcriptase, and no DNA polymerase.
• composition of embodiment 53 which further comprises deoxyribonucleotide triphosphates and optionally a buffer and/or a metal salt (e.g., magnesium chloride).
• a buffer and/or a metal salt e.g., magnesium chloride
• composition of embodiment 53 or 54 which is provided in a kit comprising the composition of embodiment 51 or 52.
• composition of embodiment 56, wherein the reagents for performing transcription further comprise ribonucleotide triphosphates and optionally a buffer and/or a metal salt (e.g., magnesium chloride).
• a metal salt e.g., magnesium chloride
• composition of embodiment 58, wherein the antibody is used in an immunoassay.
• composition of embodiment 59, wherein the immunoassay is an enzyme-linked immunosorbent assay (ELISA) or a sandwich immunoassay.
• ELISA enzyme-linked immunosorbent assay
• composition of embodiment 59 or 60, wherein the antibody used in an immunoassay comprises an unlinked antibody, an antibody bound to a solid substrate (e.g., a bead), an antibody conjugated to a detection protein or enzyme or a fragment thereof, or any combination thereof, and wherein the antibody may or may not be labeled with a dye (e.g., a fluorescent dye, a
• the one or more reagents for performing an immunoassay comprise an antibody that is specific for a target antigen or analyte
• the antibody is labeled with a dye (e.g., a fluorescent dye, a chemiluminescent dye, or a phosphorescent dye) or is conjugated to a detection protein or enzyme or a fragment thereof; and the antibody optionally is bound to a solid substrate (e.g., a bead).
• a dye e.g., a fluorescent dye, a chemiluminescent dye, or a phosphorescent dye
• a detection protein or enzyme or a fragment thereof e.g., a phosphorescent dye
• the reagents for performing a sandwich immunoassay comprise a first antibody that is specific for a target antigen or analyte and a second antibody that is specific for the target antigen or analyte; the second antibody is labeled with a dye (e.g., a fluorescent dye, a chemiluminescent dye, or a phosphorescent dye) or is conjugated to a detection protein or enzyme or a fragment thereof; and the first antibody optionally is bound to a solid substrate (e.g., a bead).
• a dye e.g., a fluorescent dye, a chemiluminescent dye, or a phosphorescent dye
• composition of embodiment 63 which comprises the first antibody and the second antibody.
• composition of embodiment 63 which comprises the first antibody and not the second antibody.
• composition of embodiment 63 which comprises the second antibody and not the first antibody.
• composition of embodiment 66 which is provided in a kit comprising the composition of embodiment 65.
• a detection antibody that is specific for a target antigen or analyte and is conjugated to a detection enzyme or a fragment thereof;
• first antibody that is specific for a target antigen or analyte
• second antibody that is specific for the first antibody and is conjugated to a detection enzyme or a fragment thereof.
• composition of embodiment 68 which comprises the detection antibody.
• composition of embodiment 68 which comprises the first antibody and the second antibody.
• composition of embodiment 68 which comprises the first antibody and not the second antibody.
• composition of embodiment 68 which comprises the second antibody and not the first antibody.
• composition of embodiment 72 which is provided in a kit comprising the composition of embodiment 71.
• composition of any one of embodiments 61 to 73, wherein the detection protein or enzyme or a fragment thereof conjugated to an antibody is selected from the group consisting of: a) phycobiliproteins, phycoerythrins, B-phycoerythrin, R-phycoerythrin, and fragments and conjugates thereof;
• peroxidases horseradish peroxidase, and fragments and conjugates thereof
• phosphatases alkaline phosphatase, and fragments and conjugates thereof.
• composition of embodiment 75, wherein the protein or enzyme useful for detection is selected from the group consisting of:
• phycobiliproteins e.g., phycoerythrins, B-phycoerythrin, R-phycoerythrin, and conjugates thereof (e.g., phycoerythrin-streptavidin conjugates, phycoerythrin-alkaline phosphatase conjugates, and phycoerythrin-antibody conjugates);
• streptavidin e.g., streptavidin-horse radish peroxidase conjugates and streptavidin-antibody conjugates
• peroxidases horseradish peroxidase, and conjugates thereof (e.g., horseradish peroxidase- antibody conjugates);
• phosphatases e.g., alkaline phosphatase- antibody conjugates.
• composition of embodiment 77, wherein the biological sample comprises whole animal (e.g., human) blood or fractionated animal (e.g., human) blood.
• composition of embodiment 77, wherein the biological sample comprises whole animal (e.g., human) plasma or fractionated animal (e.g., human) plasma.
• composition of embodiment 77 wherein the biological sample comprises whole animal (e.g., human) serum or fractionated animal (e.g., human) serum.
• the biological sample comprises animal cells, mammalian cells, human cells, plant cells, microbial cells, pathogenic cells, bacterial cells, fungal cells, protozoan cells or viral particles, or any combination thereof, or lysates or extracts thereof.
• composition of any one of the preceding embodiments which further comprises one or more substances selected from the group consisting of reducing agents, antioxidants, free radical scavengers, oxygen radical scavengers, hydroxyl radical scavengers, singlet oxygen quenchers, hydroperoxide -removing agents, protease inhibitors, nuclease inhibitors, ribonuclease (RNase) inhibitors, deoxyribonuclease (DNase) inhibitors, metal chelators, preservatives, anti-microbials, buffers (or buffering agents), detergents, and chaotropes.
• reducing agents antioxidants, free radical scavengers, oxygen radical scavengers, hydroxyl radical scavengers, singlet oxygen quenchers, hydroperoxide -removing agents, protease inhibitors, nuclease inhibitors, ribonuclease (RNase) inhibitors, deoxyribonuclease (DNase) inhibitors
• composition of embodiment 82 wherein:
• the reducing agents, the antioxidants, and the free radical scavengers are selected from the group consisting of cysteine, dithionite, dithioerythritol, dithiothreitol (DTT), dysteine, 2- mercaptoethanol, mercaptoethylene, bisulfite, sodium metabisulfite, pyrosulfite, pentaerythritol, thioglycolic acid, urea, uric acid, vitamin C, vitamin E, superoxide dismutases, and analogs, derivatives and salts thereof;
• the oxygen radical scavengers are selected from the group consisting of sugar alcohols (e.g., erythritol, mannitol, sorbitol, xylitol), monosaccharides (e.g., glucose, mannose), disaccharides (e.g., sucrose, trehalose), complex sugars, and analogs, derivatives and salts thereof;
• the hydroxyl radical scavengers are selected from the group consisting of azides, sodium azide, cysteine, dimethylsulfoxide, histidine, salicylic acid, salicylate, sugar alcohols (e.g., mannitol), monosaccharides (e.g., glucose), disaccharides (e.g., sucrose), complex sugars, and analogs, derivatives and salts thereof;
• the singlet oxygen quenchers are selected from the group consisting of azides (e.g., sodium azide), ascorbic acid, ascorbate, alkyl imidazoles (e.g., carnosine, histamine, histidine, imidazole 4- acetic acid), indoles (e.g., tryptophan and derivatives thereof, such as N-acetyl -5-methoxytryptamine, N-acetyl serotonin, 6-methoxy-l,2,3,4-tetrahydro-beta-carboline), sulfur-containing amino acids (e.g., cysteine, S-allyl-cysteine, S-aminoethyl-cysteine, djenkolic acid, ethionine, methionine, N-formyl- methionine, lanthionine, felinine), phenolic compounds (e.g., tyrosine and derivatives thereof), aromatic carboxylic acids (e.g
• the hydroperoxide-removing agents are selected from the group consisting of catalase, glutathione, peroxidases, glutathione peroxidases, pyruvate, and analogs, derivatives and salts thereof; f) the protease inhibitors are selected from the group consisting of aspartic protease inhibitors, cysteine protease inhibitors, metalloprotease inhibitors, serine protease inhibitors, threonine protease inhibitors, trypsin inhibitors (e.g., avian egg white trypsin inhibitors, bovine trypsin inhibitors, lima bean trypsin inhibitors, and soybean trypsin inhibitors such as Kunitz trypsin inhibitor and Bowman- Birk inhibitor), Kunitz-type protease inhibitors, 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF) and salts thereof (e.g., HC1 salt), amastatin, antithro
• the RNase inhibitors are selected from the group consisting of mammalian ribonuclease inhibitor proteins (e.g., porcine ribonuclease inhibitor and human ribonuclease inhibitor), aurintricarboxylic acid (ATA) and salts thereof (e.g., triammonium aurintricarboxylate (aluminon)), adenosine 5 '-pyrophosphate, 2'-cytidine monophosphate free acid (2'-CMP), 5'-diphosphoadenosine 3'-phosphate (ppA-3'-p), 5'-diphosphoadenosine 2'-phosphate (ppA-2'-p), leucine, oligovinysulfonic acid, poly(aspartic acid), tyrosine -glutamic acid polymer, 5'-phospho-2'-deoxyuridine ,3'- pyrophosphate P' ⁇ 5'-ester with adenos
• the DNase inhibitors and the metal chelators are selected from the group consisting of aurintricarboxylic acid (ATA) and salts thereof (e.g., triammonium aurintricarboxylate (aluminon)), boric acid, borate, citric acid, citrate, salicylic acid, 1 ,2-bis(o-aminophenoxy)ethane-N,N,N',N'- tetraacetic acid (BAPTA), diethylene triamine pentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), glycoletherdiaminetetraacetic acid (GEDTA), N-(2-hydroxyethyl)ethylenediamine-N,N',N'-triacetic acid (HEDTA), nitrilotriacetic acid (NTA), 2,2'- bipyridine, o-phenanthroline, triethanolamine, and analogs, a
• the preservatives are selected from the group consisting of azides (e.g., sodium azide), polyethylene glycol (PEG), and anti-microbials;
• azides e.g., sodium azide
• PEG polyethylene glycol
• anti-microbials e.g., anti-microbials
• the buffers or buffering agents are selected from the group consisting of borate, saline phosphate, saline sodium citrate, 2-(methylamino)succinic acid, N,N-bis(2-hydroxyethyl)glycine (bicine), N-tris(hydroxymethyl)methylglycine (tricine), tris(hydroxymethyl)methylamine (Tris), 2- (cyclohexylamino)ethanesulfonic acid (CHES), 4-(2-hydroxyethyl)-l -piperazineethanesulfonic acid (HEPES), piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), 2-
• TES tris(hydroxymethyl)methyl] amino ⁇ ethanesulfonic acid
• CABS 3-amino-l -propanesulfonic acid
• CABS 4-(cyclohexylamino)-l -butanesulfonic acid
• the detergents are selected from the group consisting of anionic surfactants, cationic surfactants, non-ionic surfactants, zwitterionic surfactants, ampholytic surfactants, benzethonium chloride, cetyltrimethylammonium bromide (CTAB), 3-[(3-cholamidopropyl)dimethylammonio]-l- propanesulfonate (CHAPS), 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-l- propanesulfonate (CHAPSO), N,N-dimethyl-decylamine-N-oxide, guanidinium thiocyanate, hexadecyltrimethylammonium bromide, lithium dodecyl sulfate (LDS), sodium dodecyl sulfate (SDS), sodium lauryl sulfate, sodium cholate, sodium deoxycholate, ethoxylated fatty alcohol ethers
• the chaotropes are selected from the group consisting of formamide, guanidine and salts thereof (e.g., guanidinium hydrochloride), isothiocyanate, urea, and analogs, derivatives and salts thereof.
• composition of embodiment 82 or 83, wherein the one or more substances comprise a protease inhibitor.
• composition of any one of embodiments 82 to 84, wherein the one or more substances comprise an oxygen radical scavenger.
• composition of embodiment 85 wherein the concentration of the oxygen radical scavenger by mass (mg) relative to the volume ( ⁇ .) of the at least one alcohol solvent is at least about 1%, 5%, 10%, 25%, 50%, 75%, 100%, 125% or 150%.
• composition of embodiment 86, wherein the concentration of the oxygen radical scavenger by mass (mg) relative to the volume ( ⁇ .) of the at least one alcohol solvent is about 25% to about 125%), or about 25% to about 75%.
• composition of any one of embodiments 82 to 87, wherein the one or more substances comprise:
• a metal chelator a metal chelator, a hydroxyl radical scavenger, and an RNase inhibitor
• composition of any one of the preceding embodiments which further comprises a metal salt.
• metal salt comprises an M +1 salt or an M +2 salt, or both.
• M +1 salts include lithium salts, sodium salts and potassium salts of fluoride, chloride, bromide, iodide, acetate, formate, nitrate, perchlorate (ClO f), phosphate, sulfate, tetrafluoroborate (BF 4 ⁇ ) and thiocyanate ( ⁇ SCN), and M +2 salts include magnesium salts, manganese salts and calcium salts of fluoride, chloride, bromide, iodide, acetate, formate, nitrate, perchlorate, phosphate, sulfate, tetrafluoroborate and thiocyanate.
• composition of any one of the preceding embodiments which comprises:
• ethylene glycol, 1,3-propanediol, glycerol or 1 ,2-butanediol, or any combination thereof a) ethylene glycol, 1,3-propanediol, glycerol or 1 ,2-butanediol, or any combination thereof; or b) ethylene glycol, 1,3-propanediol, glycerol or 1 ,2-butanediol, or any combination thereof, and an oxygen radical scavenger.
• composition of any one of the preceding embodiments, wherein the polypeptide, the polynucleotide or the biological sample, or any combination thereof, is soluble in the at least one alcohol solvent.
• composition of embodiment 93 wherein at least about 50%, 60%, 70%, 80%, 90% or 95% of the polypeptide, the polynucleotide or the biological sample, or any combination thereof, by mass is dissolved in the at least one alcohol solvent.
• composition of any one of the preceding embodiments, wherein the polypeptide, the polynucleotide or the biological sample, or any combination thereof, is stable after storage at a temperature from ambient temperature to about 40 °C for at least about 1 day, 3 days, 1 week,
• composition of embodiment 95 wherein the polypeptide, the polynucleotide or the biological sample, or any combination thereof, is resistant to irreversible hydrolytic damage, irreversible oxidative damage and irreversible denaturation after storage at a temperature from ambient temperature to about 40 °C for at least about 1 day, 3 days, 1 week, 2 weeks, 1 month,
• composition of embodiment 97 wherein the polypeptide, the polynucleotide or the biological sample, or any combination thereof, retain at least about 50%, 60%, 70%, 80%, 90% or 95%) of their function or activity after storage at a temperature from ambient temperature to about 40 °C for at least about 1 day, 3 days, 1 week, 2 weeks, 1 month, 3 months, 6 months or 1 year.
• a method of preserving a polypeptide, a polynucleotide or a biological sample comprising: a) mixing an aqueous mixture comprising a polypeptide, a polynucleotide or a biological sample, or any combination thereof, with at least one alcohol solvent to produce an aqueous organic mixture, wherein:
• the polypeptide comprises an enzyme that mediates a nucleic acid reaction, a polypeptide that regulates an enzyme, an antibody, a polypeptide ligand of an antibody, a polypeptide aptamer, a protein or enzyme useful for detection, a toxin, a hormone, a cytokine, a polypeptide therapeutic or a vaccine, or a derivative thereof or any combination thereof;
• the polynucleotide comprises a polynucleotide used in a nucleic acid reaction, a catalytic polynucleotide, or a polynucleotide that binds specifically to a target ligand, or a derivative thereof or any combination thereof;
• the biological sample comprises a biological fluid, a biological suspension, a fluid aspirate, blood, plasma, serum, lymph, cerebrospinal fluid, gastric fluid, bile, perspiration, ocular fluid, tears, oral fluid, sputum, saliva, a buccal sample, a tonsil sample, a nasal sample, mucus, a nasopharyngeal sample, semen, urine, a vaginal sample, a cervical sample, a rectal sample, a fecal sample, a wound or purulent sample, hair, a tissue, a tissue homogenate, cells, a cellular lysate, a tissue or cell biopsy, skin cells, tumor or cancer cells, a microbe, a pathogen, a bacterium, a fungus, a protozoan or a virus, or any combination thereof; and
• the at least one alcohol solvent is selected from the group consisting of linear and branched
• composition is in a fluid state and is substantially free of water.
• the at least one alcohol solvent in the composition comprises no more than about 20%, 10%, 5% or 2% water by mass relative to the combined mass of water and the at least one alcohol solvent.
• composition comprises one or more substances of any one of embodiments 82 to 88, and wherein the aqueous mixture, the at least one alcohol solvent or the aqueous organic mixture, or any combination thereof, comprises the one or more substances.
• composition comprises a metal salt of any one of embodiments 89 to 91, and wherein the aqueous mixture, the at least one alcohol solvent or the aqueous organic mixture, or any combination thereof, comprises the metal salt.
• aqueous solution e.g., water or an aqueous buffer
• an aqueous solution e.g., water or an aqueous buffer
• an analysis e.g., an immunoassay
• composition comprising:
• the polypeptide comprises an enzyme that mediates a nucleic acid reaction, a polypeptide that regulates an enzyme, an antibody, a polypeptide ligand of an antibody, a polypeptide aptamer, a protein or enzyme useful for detection, a toxin, a hormone, a cytokine, a polypeptide therapeutic or a vaccine, or a derivative thereof or any combination thereof;
• the polynucleotide comprises a polynucleotide used in a nucleic acid reaction, a catalytic polynucleotide, or a polynucleotide that binds specifically to a target ligand, or a derivative thereof or any combination thereof;
• the biological sample comprises a biological fluid, a biological suspension, a fluid aspirate, blood, plasma, serum, lymph, cerebrospinal fluid, gastric fluid, bile, perspiration, ocular fluid, tears, oral fluid, sputum, saliva, a buccal sample, a tonsil sample, a nasal sample, mucus, a nasopharyngeal sample, semen, urine, a vaginal sample, a cervical sample, a rectal sample, a fecal sample, a wound or purulent sample, hair, a tissue, a tissue homogenate, cells, a cellular lysate, a tissue or cell biopsy, skin cells, tumor or cancer cells, a microbe, a pathogen, a bacterium, a fungus, a protozoan or a virus, or any combination thereof; and
• composition is in a fluid state and is substantially free of water.
• composition of embodiment 112 wherein the at least one ionic organic solvent in the composition comprises no more than about 20%, 10%, 5% or 2% water by mass relative to the combined mass of water and the at least one ionic organic solvent.
• composition of embodiment 112 or 113, wherein at least about 50%, 60%, 70%, 80%, 90% or 95%) of the at least one ionic organic solvent by volume is soluble in water.
• cP centipoise
• composition of embodiment 118, wherein the at least one ionic organic solvent is a deep eutectic solvent.
• composition of any one of embodiments 112 to 119, wherein the molar ratio of the organic salt to the organic hydrogen bond donor in the at least one ionic organic solvent is from about 5: 1 to about 1 :5, or from about 3: 1 to about 1 :3, or from about 2: 1 to about 1 :2.
• the composition of embodiment 120, wherein the molar ratio of the organic salt to the organic hydrogen bond donor in the at least one ionic organic solvent is from about 1 : 1 to about 1 :2, or is about 1 : 1, about 1 : 1.5 or about 1 :2.
• composition of embodiment 122, wherein the primary ammonium salts include methylammonium salts, ethylammonium salts, propylammonium salts, butylammonium salts, 2- hydroxyethylammonium salts, 2-acetylethylammonium salts, 2-chloroethylammonium salts, 2- fluoroethylammonium salts, and benzylammonium salts.
• composition of embodiment 122 or 123, wherein the secondary ammonium salts include dimethylammonium salts, diethylammonium salts, dipropylammonium salts, dibutylammonium salts, bis(2-hydroxyethyl)ammonium salts, dibenzylammonium salts, ethylmethylammonium salts, (2- hydroxyethyl)methylammonium salts, (2-hydroxyethyl)ethylammonium salts, (2-acetylethyl)- methylammonium salts, (2-acetylethyl)ethylammonium salts, (2-chloroethyl)methylammonium salts, (2-chloroethyl)ethylammonium salts, (2-fluoroethyl)methylammonium salts, (2-fluoroethyl)ethyl- ammonium salts, benzylmethylammonium salts,
• composition of any one of embodiments 122 to 124, wherein the tertiary ammonium salts include trimethylammonium salts, triethylammonium salts, dimethylethylammonium salts, diethylmethylammonium salts, (benzyl)(ethyl)methylammonium salts, (benzyl)dimethylammonium salts, (benzyl)diethylammonium salts, (2-hydroxyethyl)(ethyl)methylammonium salts, (2- acetylethyl)(ethyl)methylammonium salts, (2-chloroethyl)(ethyl)methylammonium salts, (2- fluoroethyl)(ethyl)methylammonium salts, (2-hydroxyethyl)(benzyl)methylammonium salts,(2- hydroxyethyl)(benzyl)ethylammonium salts,
• composition of any one of embodiments 122 to 125, wherein the quaternary ammonium salts include tetramethylammonium salts, tetraethylammonium salts, (2-hydroxyethyl)trimethyl- ammonium (choline) salts, (2-hydroxyethyl)triethylammonium salts, (2-acetylethyl)trimethyl- ammonium salts, (2-acetylethyl)triethylammonium salts, (2-chloroethyl)trimethylammonium salts, (2chloroethyl)triethylammonium salts, (2-fluoroethyl)trimethylammonium salts, (2-fluoroethyl)- triethylammonium salts, (benzyl)(dimethyl)(2-hydroxyethyl)ammonium salts, (benzyl)(diethyl)(2- hydroxyethy
• composition of embodiment 127, wherein the organic salt comprises a choline salt.
• composition of any one of embodiments 122 to 128, wherein the anions of the primary ammonium salts, the secondary ammonium salts, the tertiary ammonium salts and the quaternary ammonium salts are selected from the group consisting of monovalent anions, fluoride, chloride, bromide, iodide, acetate, formate, nitrate, perchlorate (ClO ), phosphate, sulfate, tetrafluoroborate (BF 4 " ), and thiocyanate fSCN).
• composition of any one of embodiments 122 to 129, wherein the organic salt comprises choline chloride or choline acetate.
• composition of any one of embodiments 112 to 130, wherein the organic hydrogen bond donor of the at least one ionic organic solvent comprises one or more organic hydrogen bond donors selected from the group consisting of urea compounds, thiourea compounds, carbamates, amides, carboxylic acids, phenolic compounds, acyclic alcohols, and cyclic alcohols.
• composition of embodiment 131 wherein the urea compounds include urea, N- methylurea, ⁇ , ⁇ '-dimethylurea, N,N-dimethylurea, ⁇ , ⁇ , ⁇ '-trimethylurea, thiourea, N- methylthiourea, N,N'-dimethylthiourea, ⁇ , ⁇ -dimethylthiourea, and N,N,N'-trimethylthiourea.
• composition of embodiment 132, wherein the organic hydrogen bond donor comprises urea.
• composition of any one of embodiments 131 to 133, wherein the carbamates include methyl carbamate, ethyl carbamate, propyl carbamate, butyl carbamate, methyl N-methylcarbamate, ethyl N-methylcarbamate, propyl N-methylcarbamate, and butyl N-methylcarbamate.
• composition of any one of embodiments 131 to 134, wherein the amides include acetamide, propanamide, butanamide, pentanamide, benzamide, N-methylacetamide, N- methylpropanamide, N-methylbutanamide, N-methylpentanamide, and N-methylbenzamide.
• carboxylic acids include adipic acid, benzoic acid, citric acid, ethylenediaminetetraacetic acid, fumaric acid, maleic acid, malonic acid, oxalic acid, phenylacetic acid, phenylpropionic acid, propane- 1,2,3 -tricarboxylic acid (tricarballylic acid), succinic acid, and tartaric acid.
• composition of any one of embodiments 131 to 136, wherein the phenolic compounds include phenol and tyrosine.
• composition of embodiment 138, wherein the linear and branched C2-C6 acyclic alcohols having one or more hydroxyl groups are selected from the group consisting of:
• composition of embodiment 139, wherein the linear and branched C2-C5 acyclic alcohols having one hydroxyl group are selected from the group consisting of 2-chloroethanol, 2,2- dichloroethanol, 1-butanol, 1-pentanol, 2-methylbutan-l-ol, 3-methylbutanol-ol, 2,2- dimethylpropan-l-ol, 2-pentanol, 3-methylbutan-2-ol, and 3-pentanol.
• composition of embodiment 141, wherein the linear and branched C 2 -C 6 acyclic alcohols having two or three hydroxyl groups are selected from the group consisting of 1,2-ethanediol (ethylene glycol), 1 ,2-propanediol (propylene glycol), 1,3 -propanediol, 1,2,3-propanetriol (glycerol), 1 ,2-butanediol, 1,3-butanediol, 1 ,4-butanediol, 2,3-butanediol, 1,2,3-butanetriol, 1 ,2,4-butanetriol,
• composition of embodiment 142, wherein the organic hydrogen bond donor comprises ethylene glycol, 1 ,2-propanediol, 1,3-propanediol, glycerol, 1 ,2-butanediol, 1,3-butanediol, 1 ,4- butanediol, 2,3-butanediol, 1 ,2,4-butanetriol or 1,5-pentanediol, or any combination thereof.
• composition of embodiment 143, wherein the organic hydrogen bond donor comprises ethylene glycol, 1,3-propanediol, glycerol or 1,2-butanediol, or any combination thereof.
• the linear and branched C 2 - C 6 acyclic alcohols are linear and branched C 2 -C 5 acyclic alcohols having two or more hydroxyl groups.
• composition of any one of embodiments 131 to 145, wherein the cyclic alcohols include C 3 -C 6 cyclic alcohols having one or more hydroxyl groups and three to six ring carbon atoms and optionally comprising one or more halide atoms.
• composition of embodiment 146, wherein the C3-C6 cyclic alcohols having one or more hydroxyl groups are C4 or C 5 cyclic alcohols having one or more hydroxyl groups.
• composition of embodiment 147, wherein the C4 or C 5 cyclic alcohols are cyclobutanol, cyclopentanol or ascorbic acid, or any combination thereof.
• composition of any one of embodiments 112 to 150 which comprises an enzyme that mediates a nucleic acid reaction.
• composition of embodiment 151, wherein the enzyme that mediates a nucleic acid reaction comprises a topoisomerase, a helicase, a DNA polymerase, a reverse transcriptase, an RNA polymerase, a DNA ligase, an RNA ligase, a DNA repair enzyme, an RNA repair enzyme, an endonuclease, an exonuclease, a deoxyribonuclease (DNase), a ribonuclease (RNase), a transposase, a restriction enzyme or a nicking enzyme, or any combination thereof.
• DNase deoxyribonuclease
• RNase ribonuclease
• composition of embodiment 152, wherein the enzyme that mediates a nucleic acid reaction comprises a DNA polymerase, a reverse transcriptase or an RNA polymerase, or any combination thereof.
• composition of embodiment 153, wherein the enzyme that mediates a nucleic acid reaction comprises a DNA polymerase (e.g., a thermostable DNA polymerase, such as a Taq polymerase) used in polymerase chain reaction (PCR) or a reverse transcriptase used in PCR, or both.
• a DNA polymerase e.g., a thermostable DNA polymerase, such as a Taq polymerase
• PCR polymerase chain reaction
• reverse transcriptase used in PCR, or both.
• composition of embodiment 155 wherein the polynucleotide comprises at least one primer used in PCR or reverse transcription.
• the at least one primer used in PCR or reverse transcription comprises at least one pair of a forward primer and a reverse primer for amplifying at least one nucleic acid (e.g., genetic) locus, or at least one reverse transcription primer for reverse transcribing at least one polyribonucleotide, or both; and
• the at least one pair of forward primer and reverse primer optionally is labeled with a dye (e.g., a fluorescent dye) or the at least one reverse transcription primer optionally is labeled with a dye (e.g., a fluorescent dye), or both.
• a dye e.g., a fluorescent dye
• the at least one reverse transcription primer optionally is labeled with a dye (e.g., a fluorescent dye), or both.
• composition of embodiment 157, wherein the at least one primer used in PCR comprises at least 5 or 10 different pairs of forward and reverse primers for amplifying at least 5 or 10 different short tandem repeat (STR) loci utilized in a forensic database (e.g., CODIS), and
• STR short tandem repeat
• each of the at least 5 or 10 different pairs of forward and reverse primers optionally is labeled with a dye (e.g., a fluorescent dye).
• a dye e.g., a fluorescent dye
• composition of embodiment 158, wherein the at least one primer used in PCR comprises 16 different pairs of forward and reverse primers for amplifying all 13 CODIS STR loci, Penta D, Penta E and amelogenin, and
• each of the 16 different pairs of forward and reverse primers optionally is labeled with a dye (e.g., a fluorescent dye).
• a dye e.g., a fluorescent dye
• composition of any one of embodiments 112 to 159 which comprises one or more reagents for performing PCR, wherein:
• the reagents for performing PCR comprise a DNA polymerase and at least one pair of a forward primer and a reverse primer for amplifying at least one nucleic acid (e.g., genetic) locus; and the at least one pair of forward primer and reverse primer optionally is labeled with a dye (e.g., a fluorescent dye).
• a dye e.g., a fluorescent dye
• composition of embodiment 160, wherein the at least one pair of forward and reverse primers comprises at least 5 or 10 different pairs of forward and reverse primers for amplifying at least 5 or 10 different STR loci utilized in a forensic database (e.g., CODIS), and
• each of the at least 5 or 10 different pairs of forward and reverse primers optionally is labeled with a dye (e.g., a fluorescent dye).
• a dye e.g., a fluorescent dye
• composition of embodiment 161, wherein the at least one pair of forward and reverse primers comprises 16 different pairs of forward and reverse primers for amplifying all 13 CODIS STR loci, Penta D, Penta E and amelogenin, and
• each of the 16 different pairs of forward and reverse primers optionally is labeled with a dye (e.g., a fluorescent dye).
• a dye e.g., a fluorescent dye
• a thermostable DNA polymerase e.g., a Ta polymerase
• reagents for performing PCR further comprise deoxyribonucleotide triphosphates and optionally a buffer and/or a metal salt (e.g., magnesium chloride).
• composition of embodiment 164 which comprises the DNA polymerase, the at least one pair of forward and reverse primers, deoxyribonucleotide triphosphates, and optionally a buffer and/or a metal salt (e.g., magnesium chloride).
• a metal salt e.g., magnesium chloride
• composition of embodiment 164 which comprises the DNA polymerase and no primer.
• composition of embodiment 166 which further comprises deoxyribonucleotide triphosphates and optionally a buffer and/or a metal salt (e.g., magnesium chloride).
• a buffer and/or a metal salt e.g., magnesium chloride
• composition of embodiment 164 which comprises the at least one pair of forward and reverse primers and no DNA polymerase.
• composition of embodiment 168 which further comprises deoxyribonucleotide triphosphates and optionally a buffer and/or a metal salt (e.g., magnesium chloride).
• a buffer and/or a metal salt e.g., magnesium chloride
• composition of embodiment 168 or 169 which is provided in a kit comprising the composition of embodiment 166 or 167.
• the reagents for performing reverse transcription comprise a reverse transcriptase and at least one reverse transcription primer for reverse transcribing at least one polyribonucleotide;
• the at least one reverse transcription primer optionally is labeled with a dye (e.g., a fluorescent dye).
• a dye e.g., a fluorescent dye
• composition of embodiment 171, wherein the reagents for performing reverse transcription further comprise deoxyribonucleotide triphosphates and optionally a buffer and/or a metal salt (e.g., magnesium chloride).
• a metal salt e.g., magnesium chloride
• composition of embodiment 172 which comprises the reverse transcriptase, the at least one reverse transcription primer, deoxyribonucleotide triphosphates and optionally a buffer and/or a metal salt (e.g., magnesium chloride).
• a metal salt e.g., magnesium chloride
• composition of embodiment 172 which comprises the reverse transcriptase and no reverse transcription primer.
• composition of embodiment 174 which further comprises deoxyribonucleotide triphosphates and optionally a buffer and/or a metal salt (e.g., magnesium chloride).
• a buffer and/or a metal salt e.g., magnesium chloride
• composition of embodiment 172 which comprises the at least one reverse transcription primer and no reverse transcriptase.
• composition of embodiment 176 which further comprises deoxyribonucleotide triphosphates and optionally a buffer and/or a metal salt (e.g., magnesium chloride).
• composition of embodiment 176 or 177 which is provided in a kit comprising the composition of embodiment 174 or 175.
• composition of any one of embodiments 112 to 159 which comprises one or more reagents for performing reverse transcription PCR (RT-PCR), wherein:
• the reagents for performing RT-PCR comprise a reverse transcriptase, a DNA polymerase, at least one reverse transcription primer for reverse transcribing at least one polyribonucleotide to produce at least one polydeoxyribonucleotide complementary to the at least one polyribonucleotide, and at least one pair of a forward primer and a reverse primer for amplifying the at least one complementary polydeoxyribonucleotide; and
• the at least one reverse transcription primer optionally is labeled with a dye (e.g., a fluorescent dye) and the at least one pair of forward primer and reverse primer optionally is labeled with a dye (e.g., a fluorescent dye).
• a dye e.g., a fluorescent dye
• a thermostable DNA polymerase e.g., a Taq polymerase
• composition of embodiment 179 or 180, wherein the reagents for performing RT-PCR further comprise deoxyribonucleotide triphosphates and optionally a buffer and/or a metal salt (e.g., magnesium chloride).
• a buffer and/or a metal salt e.g., magnesium chloride
• composition of embodiment 181 which comprises the reverse transcriptase, the DNA polymerase, the at least one reverse transcription primer, the at least one pair of forward and reverse primers, deoxyribonucleotide triphosphates, and optionally a buffer and/or a metal salt (e.g., magnesium chloride).
• a metal salt e.g., magnesium chloride
• composition of embodiment 181 which comprises the reverse transcriptase, the DNA polymerase, no reverse transcription primer, and no pair of forward and reverse primers.
• composition of embodiment 183 which further comprises deoxyribonucleotide triphosphates and optionally a buffer and/or a metal salt (e.g., magnesium chloride).
• a buffer and/or a metal salt e.g., magnesium chloride
• composition of embodiment 185 which further comprises deoxyribonucleotide triphosphates and optionally a buffer and/or a metal salt (e.g., magnesium chloride).
• a metal salt e.g., magnesium chloride.
• composition of embodiment 188, wherein the reagents for performing transcription further comprise ribonucleotide triphosphates and optionally a buffer and/or a metal salt (e.g., magnesium chloride).
• a metal salt e.g., magnesium chloride
• composition of embodiment 190, wherein the antibody is used in an immunoassay.
• composition of embodiment 191, wherein the immunoassay is an enzyme-linked immunosorbent assay (ELISA) or a sandwich immunoassay.
• ELISA enzyme-linked immunosorbent assay
• composition of embodiment 191 or 192, wherein the antibody used in an immunoassay comprises an unlinked antibody, an antibody bound to a solid substrate (e.g., a bead), an antibody conjugated to a detection protein or enzyme or a fragment thereof, or any combination thereof, and wherein the antibody may or may not be labeled with a dye (e.g., a fluorescent dye, a
• chemiluminescent dye or a phosphorescent dye
• composition of any one of embodiments 112 to 150 which comprises one or more reagents for performing an immunoassay, wherein:
• the one or more reagents for performing an immunoassay comprise an antibody that is specific for a target antigen or analyte
• the antibody is labeled with a dye (e.g., a fluorescent dye, a chemiluminescent dye, or a phosphorescent dye) or is conjugated to a detection protein or enzyme or a fragment thereof; and the antibody optionally is bound to a solid substrate (e.g., a bead).
• a dye e.g., a fluorescent dye, a chemiluminescent dye, or a phosphorescent dye
• a detection protein or enzyme or a fragment thereof e.g., a phosphorescent dye
• composition of any one of embodiments 112 to 150 which comprises one or more reagents for performing a sandwich immunoassay, wherein:
• the reagents for performing a sandwich immunoassay comprise a first antibody that is specific for a target antigen or analyte and a second antibody that is specific for the target antigen or analyte; the second antibody is labeled with a dye (e.g., a fluorescent dye, a chemiluminescent dye, or a phosphorescent dye) or is conjugated to a detection protein or enzyme or a fragment thereof; and the first antibody optionally is bound to a solid substrate (e.g., a bead).
• a dye e.g., a fluorescent dye, a chemiluminescent dye, or a phosphorescent dye
• composition of embodiment 195 which comprises the first antibody and the second antibody.
• composition of embodiment 195 which comprises the first antibody and not the second antibody.
• composition of embodiment 195 which comprises the second antibody and not the first antibody.
• composition of embodiment 198 which is provided in a kit comprising the composition of embodiment 197.
• composition of any one of embodiments 112 to 150 which comprises one or more reagents for performing an enzyme-linked immunosorbent assay (ELISA), wherein the reagents for performing ELISA comprise:
• a detection antibody that is specific for a target antigen or analyte and is conjugated to a detection enzyme or a fragment thereof;
• first antibody that is specific for a target antigen or analyte
• second antibody that is specific for the first antibody and is conjugated to a detection enzyme or a fragment thereof.
• composition of embodiment 200 which comprises the detection antibody.
• composition of embodiment 200 which comprises the first antibody and the second antibody.
• composition of embodiment 200 which comprises the first antibody and not the second antibody.
• composition of embodiment 200 which comprises the second antibody and not the first antibody.
• composition of embodiment 204 which is provided in a kit comprising the composition of embodiment 203.
• composition of any one of embodiments 193 to 205, wherein the detection protein or enzyme or a fragment thereof conjugated to an antibody is selected from the group consisting of: a) phycobiliproteins, phycoerythrins, B-phycoerythrin, R-phycoerythrin, and fragments and conjugates thereof;
• peroxidases horseradish peroxidase, and fragments and conjugates thereof
• phosphatases alkaline phosphatase, and fragments and conjugates thereof.
• composition of embodiment 207, wherein the protein or enzyme useful for detection is selected from the group consisting of: a) phycobiliproteins, phycoerythrins, B-phycoerythrin, R-phycoerythrin, and conjugates thereof (e.g., phycoerythrin-streptavidin conjugates, phycoerythrin-alkaline phosphatase conjugates, and phycoerythrin-antibody conjugates);
• streptavidin e.g., streptavidin-horse radish peroxidase conjugates and streptavidin-antibody conjugates
• peroxidases horseradish peroxidase, and conjugates thereof (e.g., horseradish peroxidase- antibody conjugates);
• phosphatases e.g., alkaline phosphatase- antibody conjugates.
• composition of any one of embodiments 112 to 150 which comprises a biological sample, wherein the biological sample optionally can be re-hydrated by addition of water or an aqueous buffer for analysis.
• composition of embodiment 209, wherein the biological sample comprises whole animal (e.g., human) blood or fractionated animal (e.g., human) blood.
• composition of embodiment 209, wherein the biological sample comprises whole animal (e.g., human) plasma or fractionated animal (e.g., human) plasma.
• composition of embodiment 209, wherein the biological sample comprises whole animal (e.g., human) serum or fractionated animal (e.g., human) serum.
• composition of embodiment 209, wherein the biological sample comprises animal cells, mammalian cells, human cells, plant cells, microbial cells, pathogenic cells, bacterial cells, fungal cells, protozoan cells or viral particles, or any combination thereof, or lysates or extracts thereof.
• reducing agents antioxidants, free radical scavengers, oxygen radical scavengers, hydroxyl radical scavengers, singlet oxygen quenchers, hydroperoxide -removing agents, protease inhibitors, nuclease inhibitors, ribonuclease (RNase) inhibitors, deoxyribonuclease (DNase) inhibitor
• composition of embodiment 214 wherein:
• the reducing agents, the antioxidants, and the free radical scavengers are selected from the group consisting of cysteine, dithionite, dithioerythritol, dithiothreitol (DTT), dysteine, 2- mercaptoethanol, mercaptoethylene, bisulfite, sodium metabisulfite, pyrosulfite, pentaerythritol, thioglycolic acid, urea, uric acid, vitamin C, vitamin E, superoxide dismutases, and analogs, derivatives and salts thereof;
• the oxygen radical scavengers are selected from the group consisting of sugar alcohols (e.g., erythritol, mannitol, sorbitol, xylitol), monosaccharides (e.g., glucose, mannose), disaccharides (e.g., sucrose, trehalose), complex sugars, and analogs, derivatives and salt
• the hydroxyl radical scavengers are selected from the group consisting of azides, sodium azide, cysteine, dimethylsulfoxide, histidine, salicylic acid, salicylate, sugar alcohols (e.g., mannitol), monosaccharides (e.g., glucose), disaccharides (e.g., sucrose), complex sugars, and analogs, derivatives and salts thereof;
• the singlet oxygen quenchers are selected from the group consisting of azides (e.g., sodium azide), ascorbic acid, ascorbate, alkyl imidazoles (e.g., carnosine, histamine, histidine, imidazole 4- acetic acid), indoles (e.g., tryptophan and derivatives thereof, such as N-acetyl-5-methoxytryptamine, N-acetyl serotonin, 6-methoxy-l,2,3,4-tetrahydro-beta-carboline ), sulfur-containing amino acids (e.g., cysteine, S-allyl-cysteine, S-aminoethyl-cysteine, djenkolic acid, ethionine, methionine, N- formyl-methionine, lanthionine, felinine), phenolic compounds (e.g., tyrosine and derivatives thereof), aromatic carboxylic acids (e.g
• the hydroperoxide-removing agents are selected from the group consisting of catalase, glutathione, peroxidases, glutathione peroxidases, pyruvate, and analogs, derivatives and salts thereof; f) the protease inhibitors are selected from the group consisting of aspartic protease inhibitors, cysteine protease inhibitors, metalloprotease inhibitors, serine protease inhibitors, threonine protease inhibitors, trypsin inhibitors (e.g., avian egg white trypsin inhibitors, bovine trypsin inhibitors, lima bean trypsin inhibitors, and soybean trypsin inhibitors such as Kunitz trypsin inhibitor and Bowman- Birk inhibitor), Kunitz-type protease inhibitors, 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF) and salts thereof (e.g., HC1 salt), amastatin, antithro
• the RNase inhibitors are selected from the group consisting of mammalian ribonuclease inhibitor proteins (e.g., porcine ribonuclease inhibitor and human ribonuclease inhibitor),
• aurintricarboxylic acid and salts thereof (e.g., triammonium aurintricarboxylate (aluminon)), adenosine 5 '-pyrophosphate, 2'-cytidine monophosphate free acid (2'-CMP), 5'-diphosphoadenosine 3'-phosphate (ppA-3'-p), 5'-diphosphoadenosine 2'-phosphate (ppA-2'-p), leucine, oligovinysulfonic acid, poly( aspartic acid), tyrosine -glutamic acid polymer, 5'-phospho-2'-deoxyuridine 3'- pyrophosphate P' ⁇ 5'-ester with adenosine 3'-phosphate (pdUppAp), and analogs, derivatives and salts thereof;
• ATA aurintricarboxylic acid
• salts thereof e.g., triammonium aurintricarboxylate (
• the DNase inhibitors and the metal chelators are selected from the group consisting of aurintricarboxylic acid (ATA) and salts thereof (e.g., triammonium aurintricarboxylate (aluminon)), boric acid, borate, citric acid, citrate, salicylic acid, 1 ,2-bis(o-aminophenoxy)ethane-N,N,N',N'- tetraacetic acid (BAPTA), diethylene triamine pentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), glycoletherdiaminetetraacetic acid (GEDTA), N-(2-hydroxyethyl)ethylenediamine-N,N',N'-triacetic acid (HEDTA), nitrilotriacetic acid (NTA), 2,2'- bipyridine, o-phenanthroline, triethanolamine, and analogs, a
• the preservatives are selected from the group consisting of azides (e.g., sodium azide), polyethylene glycol (PEG), and anti-microbials;
• azides e.g., sodium azide
• PEG polyethylene glycol
• anti-microbials e.g., anti-microbials
• the buffers or buffering agents are selected from the group consisting of borate, saline phosphate, saline sodium citrate, 2-(methylamino)succinic acid, N,N-bis(2-hydroxyethyl)glycine (bicine), N-tris(hydroxymethyl)methylglycine (tricine), tris(hydroxymethyl)methylamine (Tris), 2- (cyclohexylamino)ethanesulfonic acid (CHES), 4-(2-hydroxyethyl)-l -piperazineethanesulfonic acid (HEPES), piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), 2-
• TES tris(hydroxymethyl)methyl] amino ⁇ ethanesulfonic acid
• CABS 3-amino-l -propanesulfonic acid
• CABS 4-(cyclohexylamino)-l -butanesulfonic acid
• the detergents are selected from the group consisting of anionic surfactants, cationic surfactants, non-ionic surfactants, zwitterionic surfactants, ampholytic surfactants, benzethonium chloride, cetyltrimethylammonium bromide (CTAB), 3-[(3-cholamidopropyl)dimethylammonio]-l- propanesulfonate (CHAPS), 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-l- propanesulfonate (CHAPSO), N,N-dimethyl-decylamine-N-oxide, guanidinium thiocyanate, hexadecyltrimethylammonium bromide, lithium dodecyl sulfate (LDS), sodium dodecyl sulfate (SDS), sodium lauryl sulfate, sodium cholate, sodium deoxycholate, ethoxylated fatty alcohol ethers
• composition of embodiment 214 or 215, wherein the one or more substances comprise a protease inhibitor.
• composition of any one of embodiments 214 to 216, wherein the one or more substances comprise an oxygen radical scavenger.
• composition of embodiment 217, wherein the concentration of the oxygen radical scavenger by mass (mg) relative to the volume ( ⁇ .) of the at least one ionic organic solvent is at least about 1%, 5%, 10%, 25%, 50%, 75%, 100%, 125% or 150%.
• composition of embodiment 218, wherein the concentration of the oxygen radical scavenger by mass (mg) relative to the volume ( ⁇ .) of the at least one ionic organic solvent is about 25% to about 125%, or about 25% to about 75%.
• composition of any one of embodiments 214 to 219, wherein the one or more substances comprise:
• a metal chelator a metal chelator, a hydroxyl radical scavenger, and an RNase inhibitor
• composition of embodiment 221 wherein the metal salt comprises an M +1 salt or an M +2 salt, or both.
• M +1 salts include lithium salts, sodium salts and potassium salts of fluoride, chloride, bromide, iodide, acetate, formate, nitrate, perchlorate (ClO ), phosphate, sulfate, tetrafluoroborate (BF 4 ⁇ ) and thiocyanate ( ⁇ SCN), and M +2 salts include magnesium salts, manganese salts and calcium salts of fluoride, chloride, bromide, iodide, acetate, formate, nitrate, perchlorate, phosphate, sulfate, tetrafluoroborate and thiocyanate.
• composition of embodiment 224 wherein at least about 50%, 60%, 70%, 80%, 90% or 95% of the polypeptide, the polynucleotide or the biological sample, or any combination thereof, by mass is dissolved in the at least one ionic organic solvent.
• composition of any one of embodiments 112 to 225, wherein the polypeptide, the polynucleotide or the biological sample, or any combination thereof, is stable after storage at a temperature from ambient temperature to about 40 °C for at least about 1 day, 3 days, 1 week,
• composition of embodiment 226, wherein the polypeptide, the polynucleotide or the biological sample, or any combination thereof, is resistant to irreversible hydrolytic damage, irreversible oxidative damage and irreversible denaturation after storage at a temperature from ambient temperature to about 40 °C for at least about 1 day, 3 days, 1 week, 2 weeks, 1 month,
• a method of preserving a polypeptide, a polynucleotide or a biological sample comprising: a) mixing an aqueous mixture comprising a polypeptide, a polynucleotide or a biological sample, or any combination thereof, with at least one ionic organic solvent to produce an aqueous organic mixture, wherein:
• the polypeptide comprises an enzyme that mediates a nucleic acid reaction, a polypeptide that regulates an enzyme, an antibody, a polypeptide ligand of an antibody, a polypeptide aptamer, a protein or enzyme useful for detection, a toxin, a hormone, a cytokine, a polypeptide therapeutic or a vaccine, or a derivative thereof or any combination thereof;
• the polynucleotide comprises a polynucleotide used in a nucleic acid reaction, a catalytic polynucleotide, or a polynucleotide that binds specifically to a target ligand, or a derivative thereof or any combination thereof;
• the biological sample comprises a biological fluid, a biological suspension, a fluid aspirate, blood, plasma, serum, lymph, cerebrospinal fluid, gastric fluid, bile, perspiration, ocular fluid, tears, oral fluid, sputum, saliva, a buccal sample, a tonsil sample, a nasal sample, mucus, a nasopharyngeal sample, semen, urine, a vaginal sample, a cervical sample, a rectal sample, a fecal sample, a wound or purulent sample, hair, a tissue, a tissue homogenate, cells, a cellular lysate, a tissue or cell biopsy, skin cells, tumor or cancer cells, a microbe, a pathogen, a bacterium, a fungus, a protozoan or a virus, or any combination thereof; and the at least one ionic organic solvent comprises an organic salt and an organic hydrogen bond donor; and
• composition is in a fluid state and is substantially free of water.
• the at least one ionic organic solvent in the composition comprises no more than about 20%, 10%, 5% or 2% water by mass relative to the combined mass of water and the at least one ionic organic solvent.
• composition comprises one or more substances of any one of embodiments 214 to 220, and wherein the aqueous mixture, the at least one ionic organic solvent or the aqueous organic mixture, or any combination thereof, comprises the one or more substances.
• a metal chelator a hydroxyl radical scavenger, and an RNase inhibitor
• a hydroxyl radical scavenger and a DNase inhibitor a metal chelator, a hydroxyl radical scavenger, and an RNase inhibitor
• a hydroxyl radical scavenger and a DNase inhibitor a metal chelator, a hydroxyl radical scavenger, and an RNase inhibitor
• composition comprises a metal salt of any one of embodiments 221 to 223, and wherein the aqueous mixture, the at least one ionic organic solvent or the aqueous organic mixture, or any combination thereof, comprises the metal salt.
• an aqueous solution e.g., water or an aqueous buffer
• a biochemical reaction e.g., PCR
• an analysis e.g., an immunoassay
• a container comprising the composition of any one of embodiments 1 to 98 and 112 to 229.
• the container of embodiment 243 which is a tube, a vial, a well or a chamber (including a well or chamber in a cartridge).
• kits comprising the composition of any one of embodiments 1 to 98 and 112 to 229.
• kit of embodiment 245 which comprises reagents for performing a biochemical reaction (e.g., a nucleic acid amplification reaction) or an assay (e.g., an immunoassay), wherein the kit contains:
• composition comprising the reagents for performing the biochemical reaction or the assay
• compositions comprising the reagents for performing the biochemical reaction or the assay.
• aqueous solution e.g., water or an aqueous buffer
• kit of any one of embodiments 245 to 247, which further comprises a desiccant which further comprises a desiccant.
• kit of any one of embodiments 245 to 248, which further comprises instruction for storing and using a composition, and optionally instruction for using an aqueous solution (e.g., water or an aqueous buffer) to re-hydrate the polypeptide, the polynucleotide or the biological sample, or any combination thereof, of the composition.
• aqueous solution e.g., water or an aqueous buffer
• composition comprising:
• composition is in a fluid state and is substantially free of water (e.g., comprising no more than about 10%, 5% or 2% water by mass relative to the combined mass of water and the composition).
• composition of embodiment 250 comprising a DNA polymerase (e.g., a thermostable DNA polymerase) and deoxyribonucleotide triphosphates.
• a DNA polymerase e.g., a thermostable DNA polymerase
• deoxyribonucleotide triphosphates e.g., a thermostable DNA polymerase
• composition of embodiment 255 further comprising at least one reverse transcription primer for reverse transcribing at least one polyribonucleotide sequence.
• composition of embodiment 250 comprising reagents (e.g., sufficient) for performing PCR or reverse transcription PCR on a polynucleotide analyte (e.g., DNA and/or RNA), except for at least one pair of forward and reverse PCR primers and/or at least one reverse transcription primer.
• reagents e.g., sufficient
• a polynucleotide analyte e.g., DNA and/or RNA
• composition of embodiment 250 comprising reagents (e.g., sufficient) for performing PCR or reverse transcription PCR on a polynucleotide analyte (e.g., DNA and/or RNA), including at least one pair of forward and reverse PCR primers and/or at least one reverse transcription primer.
• reagents e.g., sufficient
• a polynucleotide analyte e.g., DNA and/or RNA
• composition of embodiment 250 comprising a first antibody against a first target analyte, and optionally a second, different antibody against the first antibody, the first target analyte, or a second, different target analyte, wherein the first antibody and/or the optional second antibody can be labeled with a dye (e.g., a fluorescent dye) or can be conjugated to a detection protein or enzyme or a fragment thereof.
• a dye e.g., a fluorescent dye
• composition of embodiment 250 comprising reagents (e.g., sufficient) for performing an immunoassay on a target analyte.
• composition of embodiment 260, wherein the immunoassay comprises an enzyme-linked immunosorbent assay (ELISA) or a sandwich immunoassay.
• ELISA enzyme-linked immunosorbent assay
• a method comprising: providing a composition comprising:
• At least one C 2 -C 6 alcohol solvent having one or more hydroxyl groups wherein the at least one alcohol solvent is substantially soluble in water (e.g., at least about 90% soluble in water by volume or miscible in water) and has a boiling point substantially greater than that of water (e.g., a boiling point of at least about 110 °C, 125 °C or 150 °C at a pressure of about 1 atm);
• composition is in a fluid state and is substantially free of water (e.g., comprising no more than about 10%, 5% or 2% water by mass relative to the combined mass of water and the composition); and
• aqueous solution e.g., water or an aqueous buffer.
• the composition comprises reagents (e.g., sufficient) for performing PCR or reverse transcription PCR on a polynucleotide analyte (e.g., DNA and/or RNA), except for at least one pair of forward and reverse PCR primers and/or at least one reverse transcription primer; and
• the method comprises contacting the rehydrated composition with a polynucleotide analyte (e.g., DNA and/or RNA) and with at least one pair of forward and reverse PCR primers and/or at least one reverse transcription primer.
• a polynucleotide analyte e.g., DNA and/or RNA
• the composition comprises reagents (e.g., sufficient) for performing PCR or reverse transcription PCR on a polynucleotide analyte (e.g., DNA and/or RNA), including at least one pair of forward and reverse PCR primers and/or at least one reverse transcription primer; and
• the method comprises contacting the rehydrated composition with a polynucleotide analyte (e.g., DNA and/or RNA).
• a polynucleotide analyte e.g., DNA and/or RNA
• the composition comprises reagents (e.g., sufficient) for performing an immunoassay (e.g., an ELISA or a sandwich immunoassay) on a target analyte; and
• an immunoassay e.g., an ELISA or a sandwich immunoassay
• the method comprises contacting the rehydrated composition with a target analyte.
• kits comprising a first container containing a composition, wherein the composition comprises:
• composition is in a fluid state and is substantially free of water (e.g., comprising no more than about 10%, 5% or 2% water by mass relative to the combined mass of water and the composition).
• reagents e.g., sufficient for performing PCR on a polynucleotide analyte, optionally excluding or including at least one pair of forward and reverse PCR primers;
• reagents e.g., sufficient for performing reverse transcription PCR on a polynucleotide analyte, optionally excluding or including at least one pair of forward and reverse PCR primers and/or at least one reverse transcription primer;
• reagents e.g., sufficient for performing an immunoassay (e.g., an ELISA or a sandwich immunoassay) on a target analyte.
• an immunoassay e.g., an ELISA or a sandwich immunoassay
• a DNA polymerase e.g., a thermostable DNA polymerase
• nucleotide triphosphates e.g., deoxyribonucleotide triphosphates
• optionally a buffer, a monovalent or divalent metal salt (e.g., magnesium chloride), and/or at least one pair of forward and reverse PCR primers e.g., magnesium chloride
• a DNA polymerase e.g., a thermostable DNA polymerase
• a reverse transcriptase and nucleotide triphosphates e.g., deoxyribonucleotide triphosphates
• a buffer e.g., a monovalent or divalent metal salt (e.g., magnesium chloride), at least one pair of forward and reverse PCR primers, and/or at least one
• the reagents (e.g., sufficient) for performing an immunoassay on a target analyte comprise at least one antibody against a target analyte.
• kit of any one of embodiments 267 to 269 further comprising a second container containing an aqueous solution (e.g., water or an aqueous buffer) for rehydrating the composition.
• aqueous solution e.g., water or an aqueous buffer
• composition of any of embodiments 1-3 which comprises one or more reagents for performing DNA processing such as library preparation for next generation sequencing of DNA, wherein: one reagent for performing DNA processing comprises a T4 polymerase or an equivalent polymerase and the composition further comprises dNTPs to generate blunt ended DNA fragments as would be required for next generation sequencing library preparation.
• composition of any of embodiments 1-3 which comprises one or more reagents for performing DNA processing such as library preparation for next generation sequencing of DNA, wherein: one reagent performing DNA processing comprises a T4 polynucleotide kinase or an equivalent kinase and the composition further comprises ATP to phosphorylate blunt ended DNA.
• composition of any of embodiments 1-3 which comprises one or more reagents for performing DNA processing such as library preparation for next generation sequencing of DNA, wherein: one reagent performing DNA processing comprises a T4 ligase or an equivalent ligase and the composition further comprises ATP to ligate adapters onto the phosphorylated, blunt ended DNA.
• composition of any of embodiments 1-3 which comprises one or more reagents for performing DNA processing such as library preparation for next generation sequencing of DNA, wherein: one reagent performing DNA processing comprises a Klenow polymerase or an equivent DNA polymerase and the composition further comprises dATP to generate a poly-A tail onto the phosphorylated, blunt ended DNA.
• composition of any of embodiments 1-3 which comprises one or more reagents for performing DNA processing such as library preparation for next generation sequencing of DNA, wherein: one reagent performing DNA processing comprises a T4 polymerase or an equivalent polymerase to generate blunt ended DNA fragments.
• composition of any of embodiments 1-3 which comprises one or more reagents for performing DNA processing such as library preparation for next generation sequencing of DNA, wherein: one reagent performing DNA processing comprises a T4 polynucleotide kinase or an equivalent kinase and ATP to phosphorylate blunt ended DNA.
• composition of any of embodiments 1-3 which comprises one or more reagents for performing DNA processing such as library preparation for next generation sequencing of DNA, wherein: one reagent comprising a T4 ligase or an equivalent ligase and ATP to ligate adapters onto the phosphorylated, blunt ended DNA.
• composition of any of embodiments 1-3 which comprises one or more reagents for performing DNA processing such as library preparation for next generation sequencing of DNA, wherein: one reagent performing DNA processing comprises a Klenow polymerase or an equivalent DNA polymerase and dATP to generate a poly-A tail onto the phosphorylated, blunt ended DNA.
• composition of embodiment 151 wherein the enzyme that mediates a nucleic acid reaction comprises a T4 polymerase or an equivalent polymerase and the composition further comprises dNTPs to generate blunt ended DNA fragments as would be required for next generation sequencing library preparation.
• the enzyme that mediates a nucleic acid reaction comprises a T4 polynucleotide kinase or an equivalent kinase and the composition further comprises ATP to phosphorylate blunt ended DNA as would be required for next generation sequencing library preparation.
• composition of embodiment 151 wherein the enzyme that mediates a nucleic acid reaction comprises a T4 ligase or an equivalent ligase and the composition further comprises ATP to ligate adapters onto the phosphorylated, blunt ended DNA as would be required for next generation sequencing library preparation.
• composition of embodiment 151 wherein the enzyme that mediates a nucleic acid reaction comprises a Klenow polymerase or an equivent DNA polymerase and the composition further comprises dATP to generate a poly-A tail onto the phosphorylated, blunt ended DNA as would be required for next generation sequencing library preparation.
• the enzyme that mediates a nucleic acid reaction comprises a Klenow polymerase or an equivent DNA polymerase and the composition further comprises dATP to generate a poly-A tail onto the phosphorylated, blunt ended DNA as would be required for next generation sequencing library preparation.
• the evaporated mixture containing the RT-PCR reagents and optionally sucrose in glycerol was stored in the PCR tube capped with a snap-on cap at ambient temperature (about 25 °C) for varying periods of time (about 0 day, 7 days or 14 days).
• nuclease-free water and template RNA (about 10 ng of HeLa total RNA) were added to the evaporated mixture in the PCR tube to a final volume of about 25 ⁇ L.
• Reverse transcription PCR was performed according to the manufacturer's recommended protocol.
• Production of the target 18S rRNA amplicon product having about 313 base pairs (bp) was analyzed by gel electrophoresis using 2% agarose gel and UV visualization.
• Positive control was the Master Mix not treated with sucrose and not subjected to water removal, where the Master Mix was prepared by taking unmixed RT-PCR reagents out of a -20 °C freezer and mixing them shortly before use at all tested time points (untreated and unevaporated Master Mix), with addition of the template RNA. Negative control was untreated and unevaporated Master Mix without addition of the template RNA.
• the target 313 bp 18S rRNA product was produced if no sucrose or about 1.3 mg, 2.5 mg or 5 mg of sucrose had been added to the RT-PCR Master Mix.
• AIV-M Avian Influenza Virus
• RNA was placed in a well of a 96- well optical PCR plate.
• the volume of Master Mix contained some amount (e.g., about 0.5-4 ⁇ ,) of glycerol. Varying amounts of sucrose (0 mg, or about 1.3 mg or 2.5 mg) were added to the Master Mix. Water was removed from the resulting mixture by evaporation at reduced pressure (about 0.2 atm) and ambient temperature overnight to yield a fluid mixture having a volume of about 4 ⁇ ⁇ .
• the evaporated mixture containing the RT-PCR reagents and optionally sucrose in glycerol was stored in the well capped with a snap-on cap at ambient temperature (about 25 °C) for varying periods of time (about 0 day or 7 days).
• the PCR plate was kept in the dark during storage to protect a dye-labeled probe in the RT-PCR reagents from light.
• avian influenza virus matrix RNA For analysis of avian influenza virus matrix RNA, deionized nuclease-free water and template RNA (about 10 ng of AIV genomic RNA) were added to the evaporated mixture in the well to a final volume of about 25 ⁇ ⁇ . Reverse transcription PCR was performed according to the manufacturer's recommended protocol. Production of the target amplicon product of the matrix (M) gene of AIV was analyzed using an Applied Biosystems 7500 Fast Real-Time PCR System (Life Technologies), which generated Ct values when a fluorescent signal reached a level detectable by the real-time PCR instrument.
• Applied Biosystems 7500 Fast Real-Time PCR System (Life Technologies), which generated Ct values when a fluorescent signal reached a level detectable by the real-time PCR instrument.
• Positive control was the Master Mix not treated with sucrose and not subjected to water removal, where the Master Mix was prepared by taking unmixed RT-PCR reagents out of a -20 °C freezer and mixing them shortly before use at all tested time points (untreated and unevaporated Master Mix), with addition of the template RNA. Negative control was untreated and unevaporated Master Mix without addition of the template RNA.
• addition of no sucrose to the RT-PCR Master Mix yielded a slightly lower amount of the target matrix product compared to addition of about 1.3 mg or 2.5 mg of sucrose to the Master Mix.
• addition of no sucrose to the Master Mix resulted in no detectable target matrix product, while addition of about 1.3 mg of sucrose to the Master Mix produced a lower amount of the target matrix product than addition of about 2.5 mg of sucrose to the Master Mix.
• An undiluted volume (about 20 ⁇ .) of the Master Mix of the PowerPlex ® 16 HS System (Promega) for human identification was placed in a PCR tube.
• the volume of Master Mix contained some amount (e.g., about 0.5-4 ⁇ .) of glycerol.
• the Master Mix contained 16 different pairs of forward and reverse primers for amplifying all 13 current CODIS STR loci, as well as Penta D, Penta E and amelogenin. Each primer was labeled with a fluorescent dye, and a total of three different fluorescent dyes were used to label the 16 different pairs of forward and reverse primers. Varying amounts of sucrose (0 mg, or about 1 mg or 2 mg) were added to the Master Mix.
• Positive control was the Master Mix not treated with sucrose and not subjected to water removal, where the Master Mix was prepared by taking unmixed PCR reagents out of a -20 °C freezer and mixing them shortly before use at all tested time points (untreated and unevaporated Master Mix), with addition of the template DNA. Negative control was untreated and unevaporated Master Mix without addition of the template DNA.
• addition of no sucrose or about 1 mg or 2 mg of sucrose to the PCR Master Mix yielded similar amounts of the amplicon products of the 16 loci ( Figure 2).
• aqueous solution containing varying amounts of glycerol (0 mg, or about 2.5 mg, 5 mg, 7.5 mg or 10 mg) and an aqueous solution containing varying amounts of sucrose (0 mg, or about 5 mg, 7.5 mg or 10 mg) were added to a volume (about 50 ⁇ 3 ⁇ 4 of human serum in natural serum fluid in a tube.
• the volume of human serum contained about 7.5 mg of serum solids (non- volatile components of serum). Water was removed from the resulting mixture by evaporation at reduced pressure (about 0.2 atm) and ambient temperature for about two days.
• Figure 4 shows evaporated mixtures containing about 7.5 mg of human serum solids and varying amounts of glycerol and sucrose, and the approximate volume of the mixtures after evaporation of water at about 0.2 atm and ambient temperature for about two days.
• the five evaporated mixtures containing human serum solids and sucrose in glycerol were clear and showed no apparent flocculation or precipitation of serum solids.
• the evaporated mixture containing the RT-PCR reagents and sucrose, and optionally mineral oil or a detergent, in glycerol or glycerol/1, 3 -propanediol was rehydrated for immediate testing or was stored in the PCR tube capped with a snap-on cap at ambient temperature (about 25 °C) for about 7 days before rehydration and testing.
• nuclease-free water and template RNA (about 10 ng of HeLa total RNA) were added to the evaporated mixture in the PCR tube to a final volume of about 25 ⁇ L.
• Reverse transcription PCR was performed according to the manufacturer's recommended protocol.
• Production of the target 18S rRNA amplicon product having about 313 base pairs (bp) was analyzed by gel electrophoresis using 2% agarose gel and ethidium bromide for UV visualization.
• 1,3-PD 1,3 -propanediol
• No overlay no mineral oil and no detergent had been added to the Master Mix.
• Mineral oil mineral oil, but no detergent, had been added to the Master Mix, and the mineral oil was removed before RT-PCR was conducted.
• For the "1% Triton-X” tests 1% Triton®-X, but no mineral oil, had been added to the Master Mix.
• a Luminex MagPix bead-based device is used to perform immunoassays.
• the MagPix bead based immunassay technology is used to demonstrate preservation human immunoglobulin reagents in high boiling point alcohol solvents at the extremes of temperature.
• a commercial (triplex) human immunoglobulin immunoassay capable of quantifying human immunoglobulins IgA, IgGl, IgM in parallel is used, in the Luminex MagPix bead format.
• a highly purified immunoglobulin standard cocktail is employed, comprising purified human [IgA, IgGl, IgM] at a fixed ratio.
• the sample is re-hydrated by addition of 50uL of water, mixed by pipetting, then subjected to a series of dilutions, prior to addition of anti-Ig antibody-coated magnetic beads, standard solution phase binding analysis and finally, analysis via bead imaging on the Luminex MagPix device.
• sucrose 5mg
• HPBCD Hydrophilicity-Bassham
• Fig 6a describes the effect of 5hrs of dry, storage at 25C in several high boiling point alcohol solvents.
• a number of solvent treatment formulations display Ig analyte concentrations which are within 20% of the always frozen control Ig standards (green arrow).
• those high boiling point alcohol combinations which show the lowest apparent recovery of Ig analytes are those which lack sucrose or HPBCD as antioxidant stabilizers added to the high boiling point alcohol solvents.
• This example shows the preservation of human serum in high boiling point (poly) alcohol solvents at ambient temperature upon removal of water.
• Serum concentration during desiccation is minimized by adding a volume of high boiling point alcohol solvent which is identical to the volume of the serum to be preserved. The result of this is that, subsequent to water removal by evaporation at ambient temperature, serum analyte concentration in the resulting water-free high boiling point alcohol solvent or solvent pair is the same as that of the original serum starting material.
• Example 8 Table A. Influenza q-rtPCR Test Developed
• Example 8 Table C. Stabilization of a q-rtPCR Reaction Kit for Human Influenza:
• Example 8 show that the [Glycerol: 1,3PD] solvent pair [G:PD] alone or with sucrose added as an antioxidant and stabilizer preserves q-rtPCR reaction components upon desiccation to a substantially water free fluid state.
• An undiluted volume (about 20 ⁇ .) of the Master Mix of the PowerPlex ® 16 HS System (Promega) for human identification was placed in a PCR tube.
• the volume of Master Mix contained some amount (e.g., about 0.5-4 mg) of glycerol to which an additional O. lmg of glycerol was added . To that was added an additional 0.5mg of sorbitol or sucrose.
• the Master Mix contained 16 different pairs of forward and reverse primers for amplifying all 13 current CODIS STR loci, as well as Penta D, Penta E and amelogenin.
• Each primer was labeled with a fluorescent dye, and a total of three different fluorescent dyes were used to label the 16 different pairs of forward and reverse primers.
• Water was removed from the resulting mixture by evaporation at reduced pressure (about 0.2 atm) and ambient temperature overnight to yield a fluid mixture having a volume of about 4 ⁇ .
• the evaporated mixture containing the PCR reagents and optionally sucrose in glycerol was stored in the PCR tube capped with a snap-on cap at 37 °C for one week. The PCR tube was kept in the dark during storage to protect the dye-labeled primers from light.

Landscapes

• Life Sciences & Earth Sciences (AREA)
• Health & Medical Sciences (AREA)
• Engineering & Computer Science (AREA)
• Dentistry (AREA)
• General Health & Medical Sciences (AREA)
• Wood Science & Technology (AREA)
• Zoology (AREA)
• Environmental Sciences (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Applications Claiming Priority (6)

Publications (1)

Family

ID=50435493

Family Applications (1)

Country Status (1)

Cited By (37)

Citations (1)

• 2013
  • 2013-10-04 WO PCT/US2013/063558 patent/WO2014055936A1/fr not_active Ceased

Patent Citations (1)

Non-Patent Citations (3)

Similar Documents

Legal Events