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WO2011068518A1 - Analyse en point final d'une pcr quantitative multiplexée d'acides nucléiques cibles - Google Patents

Analyse en point final d'une pcr quantitative multiplexée d'acides nucléiques cibles Download PDF

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WO2011068518A1
WO2011068518A1 PCT/US2009/066778 US2009066778W WO2011068518A1 WO 2011068518 A1 WO2011068518 A1 WO 2011068518A1 US 2009066778 W US2009066778 W US 2009066778W WO 2011068518 A1 WO2011068518 A1 WO 2011068518A1
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nucleic acid
primer
particles
labeled
probes
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Daniel Colin Pregibon
Patrick Seamus Doyle
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Massachusetts Institute of Technology
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Massachusetts Institute of Technology
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification

Definitions

  • This invention generally relates to methods for performing quantitative multiplexed endpoint PCR in a single chamber to amplify, detect and quantify the amount of one or more target nucleic acids in a complex sample using encoded polymer particles on which are immobilized probes that are complementary to the targets.
  • Certain embodiments of the invention are directed to a method of amplifying and quantifying a plurality of nucleic acid targets in a sample having the steps of: a) combining in a chamber: the sample comprising the plurality of nucleic acid targets; a labeling agent, a plurality of primer pairs for priming amplification of the plurality of nucleic acid targets, wherein the primer pairs hybridize to the targets at a primer annealing temperature, a plurality of particles on which are immobilized a plurality of nucleic acid probes that are complementary to the plurality of nucleic acid targets, and a PCR cocktail containing enzymes for amplifying the nucleic acid targets; b.) performing one or more amplification cycles to form labeled amplification products for each of the plurality of nucleic acid targets, c.) hybridizing the labeled amplification products for each of the plurality of nucleic acid targets to the respective complementary probes at a hybridization temperature that is at least from about 2-15
  • amplification product signal for each of the nucleic acid targets to a signal from a known amount of a known reference nucleic acid to quantify the amount of each nucleic acid target in the sample.
  • the known reference nucleic acid of step e) is an endogenous reference gene (such as nucleolar RNA, beta-actin, GADPH or 18S R A), or an external nucleic acid added to the sample, or the nucleic acid target known amounts of which are plotted on a standard curve.
  • the labeling agent binds to one primer of each of the primer pairs.
  • the mixture of step a further includes a free probe that is complementary to a region of the amplification product and wherein the free probe is bound to the labeling agent such as a fluorescent label selected from the group comprising 6-FAMTM, Alexa Fluor,
  • the probe includes a 10 base pair to about 50 base pair sequence that is complementary to the target nucleic acid sequence and the 3' end of the probe includes a blocked 3' hydroxyl group such as a phosphate group or a 3' inverted dT or dideoxycytidine modification.
  • the probes include a locked nucleic acid (LNA) modification.
  • the primer annealing temperature is from about 35 °C to about 60 °C and the hybridization temperature is from about 37 to about 75 °C. In other embodiments the primers are from about 10 to about 25 base pairs in length and the
  • amplification products are from about 50 to about 100 base pairs long, preferably from about 50 to about 70 base pairs long.
  • the primers have a melting temperature of about 35 °C to about 65 °C and the probe-amp licon complexes have a melting temperature of about 40 to about 75 °C.
  • the hybridization step (c) is from about 20 minutes to about 90 minutes long and from about 20 to about 40 amplification cycles are performed in step (b).
  • the particles are encoded polymer particles preferably hydrogels of polyethylene glycol, that include one or more different probes that are either immobilized on the particle or incorporated into the particle substrate.
  • the particles can be encoded using fluorophores, chromophores, graphical codes, radio frequencies, magnetic properties, radioactivity, or diffractive gratings, and they can be composed of polymer, glass, silica, or metal.
  • An embodiment is directed to a method of amplifying and detecting genetic variation such as an SNP at a known mutation site in a gene in a sample comprising a plurality of nucleic acid targets having the steps of: a) combining in a chamber: a first primer pair for priming amplification of a first allele of the gene using multiplex PCR, wherein the primers hybridize to the targets at a primer annealing temperature, and wherein one primer of the first primer pair is labeled with a first reporter, and a second primer pair for priming amplification of a second allele of the gene using multiplex PCR, wherein the primers hybridize to the targets at a primer annealing temperature, and wherein one primer of the first primer pair is labeled with a second reporter, a plurality of particles on which are immobilized a plurality of nucleic acid probes that are complementary to a nucleic acid sequence that is common to both the first and second alleles, wherein the common sequence is adjacent to
  • the reporter can be any reporter known in the art including fluorophores, quantum dots, and radioisotopes.
  • the primer labeled with the first reporter includes a nucleotide at the 3 ' end that is complementary to the nucleotide in the first allele at the known mutation site
  • the primer labeled with the second reporter includes a nucleotide at the 3 ' end that is complementary to the nucleotide in the second allele at the known mutation site
  • the nucleic acid probe that is complementary to a nucleic acid sequence that is common to two different alleles of a particular gene, for example the sequence is adjacent to a known SNP mutation site on the gene. Certain embodiments are directed to this probe, and other
  • embodiments are directed to a particle for nucleic acid detection, that includes the probe.
  • kits for detecting genetic variation at a known mutation site in a gene that include a first allele-specific primer pair for priming amplification of a first allele of the gene, wherein the primer that is extended to form the amplification product is labeled with a first reporter, and a second allele-specific primer pair for priming amplification of a second allele of the gene, wherein the primer that is extended to form the amplification product is labeled with a second reporter, and encoded hydrogel particles on which are immobilized a plurality of nucleic acid probes that are complementary to a nucleic acid sequence that is common to both the first and second alleles, wherein the common sequence is adjacent to the known mutation site.
  • FIG. 1 Encoded particle assay limits of detection vs. time.
  • 1 A Sensitivities were identified as the point where the measured signal-to-noise ratio was 3, and were plotted against a quantitative model.
  • FIG. IB Specificity was confirmed using four microRNA let-7 family members. Let-7a synthetic target was spiked into E. coli RNA together with particles bearing probe regions complementary to let7a, 7b, 7c, and 7d, respectively, which have sequences differing by only one or two nucleotides.
  • FIG. 2 Demonstration of thermal stability of encoded particles and proof of no-rinse assay. Hydrogel particles bearing two probe regions, a negative control region, and barcode region were heated at 95°C for 20 min. They were then incubated with 500 attomole target at 37°C for 60 minutes and imaged immediately afterward. No rinsing steps were used prior to scanning to detect fluorescence. Scale bar is 50 ⁇ .
  • FIG. 3 Multiplexed End Point-PCR for multiplexed detection of nucleic acid targets from lambda phage. Fluorescence images of particles after PCR. Shown is a schematic of the particle design, and an image of fluorescence on scanned particles hybridized to labeled amplicons from two samples containing lambda phage DNA and either primers for target #1 or target #2.
  • FIG. 4 Multiplexed PCR detection and quantification of mRNA amplicons.
  • Four different mRNA targets and an internal reference were reverse-transcribed and amplified using RT-PCR.
  • Targets detected with multiplexed encoded hydrogel particles ("Barcoded particles") compared to Luminex particles.
  • FIG. 5 Schematic of a method for amplifying and detecting pathogens in a complex nucleic acid sample.
  • Allele-specific PCR is a technique that is used to identify point mutations called single-nucleotide polymorphisms (SNPs) (single base differences in DNA). It requires prior knowledge of a DNA sequence, including differences between alleles, and uses primers whose 3' ends encompass the point mutation.
  • SNPs single-nucleotide polymorphisms
  • Asymmetric PCR means the preferential amplification of one strand of the original DNA more than the other. PCR is carried out as usual, but with a great excess of the primers for the chosen strand.
  • Annealing in genetics, means for DNA or RNA to pair by hydrogen bonds to a complementary sequence, forming a double-stranded polynucleotide.
  • the term is often used to describe the binding of a DNA probe or of a primer to a DNA strand during a polymerase chain reaction (PCR).
  • Encoded Particle means a polymer particle, preferably a hydrogel, which is labeled with an identifying characteristic (one or multiple fluorescent or features, a graphical pattern, magnetic properties, a radio frequency tag, etc.) and has a nucleic acid probe
  • Encoded particles preferably have a fluorescently labeled coded region and at least one distinct probe region comprising a plurality of nucleic acid probes that hybridize to a particular target nucleic acid.
  • the encoded particles can have more than one distinct probe region that are each specific for a different particular target nucleic acid.
  • the encoded particles also has an inert region that is not labeled, separating the coded region from the first flanking probe region.
  • Hybridization temperature as used herein means the temperature at which the PCR hybridization step is conducted during which step the PCR-generated amplification products hybridize to the corresponding complementary probes immobilized on the particles.
  • Locked nucleic acid or "LNA” means a bi-cyclic compound that is structurally similar to RNA nucleosides. LNAs have a furanose conformation that is restricted by a methylene linker that connects the 2'-0 position to the 4'-C position. Locked nucleic acids can increase complex stability approximately tenfold and can alter the hybridization temperature of a nucleic acid to a probe.
  • T m Melting Temperature
  • Multiplex -PCR means the use of multiple, unique primer sets within a single PCR mixture to produce amplicons of varying sizes specific to different DNA sequences.
  • Multiplexed End Point Quantitative PCR means a one pot multiplexed PCR method for amplification and end point detection and quantification of multiple nucleic acid targets in a single sample, wherein a complex sample is combined with primer sets unique for each targeted nucleic acid, PCR cocktail, enzymes, labeling agent (which can be free or attached to a primer for example) and a plurality of particles, preferably encoded polymer particles, on which are immobilized a plurality of probes complementary to each respective nucleic acid targets.
  • the method has only one cycle of amplification followed by a single hybridization step, preferably at a hybridization temperature that is at least about 2 to about 15 °C higher than the primer annealing temperature.
  • the signal from the labeled amplification product bound to the corresponding complementary probes on the particle is detected and quantified.
  • the amplification product signal is then compared to either 1) a standard curve that correlates the signal generated by binding of a nucleic acid target to the corresponding probe over a broad range of target concentrations, 2) an endogenous reference gene or 3) an external target that is spiked into the sample at a known concentration to quantitate the amount of nucleic acid target in the complex sample.
  • PCR cocktail as used herein means a solution for PCR including: deoxynucleotide triphosphates (dNTPs); a buffer solution providing a suitable chemical environment for optimum activity and stability of the DNA polymerase and/or reverse transcriptase; divalent cations, typically magnesium ions (Mg +); and monovalent cation potassium ions.
  • dNTPs deoxynucleotide triphosphates
  • buffer solution providing a suitable chemical environment for optimum activity and stability of the DNA polymerase and/or reverse transcriptase
  • divalent cations typically magnesium ions (Mg +); and monovalent cation potassium ions.
  • Primer annealing temperature means the temperature at which a primer anneals to a DNA target that is to be amplified.
  • the primer melting temperature is the estimate of the DNA- DNA hybrid stability and it is critical in determining the annealing temperature.
  • Reverse Transcription PCR means a method used to amplify, isolate or identify a known RNA sequence from a sample such as cellular or tissue RNA. The PCR is preceded by a reaction using reverse transcriptase to convert RNA to cDNA. RT-PCR is widely used in expression profiling, to determine the expression of a gene or to identify the sequence of an RNA transcript, including transcription start and termination sites. The present methods are suitable for RT-PCR of RNA targets.
  • Certain embodiments of the present invention are directed to one pot multiplexed quantitative PCR methods for end point analysis of a plurality of nucleic acid targets in a complex sample without user intervention.
  • a complex sample is combined with primer sets unique for each targeted nucleic acid, PCR cocktail, enzymes, labeling agent (which can be free or attached to a primer, for example) and a plurality of particles, preferably encoded polymer particles with one or more probe regions on which are immobilized a plurality of probes complementary to each respective nucleic acid target.
  • the method has only one set of amplification cycles followed by a single hybridization step at a hybridization temperature that is significantly higher than the primer annealing temperature, preferably at least about 2 to about 15 °C higher.
  • the signal from the labeled amplification products bound to the corresponding complementary probes on the particle is detected and quantified. Typically about 30 amplification cycles are needed to generate the sufficient amount of labeled amplicons for detection.
  • Any particles on which a nucleic acid probe can be immobilized can be used; however, encoded polymer particles made with a porous hydrogel matrix in which the probes are dispersed are preferred. These particles have an encoded region and one or more distinct probe regions in which are immobilized a plurality of probes that are complementary to a particular target nucleic acid.
  • encoded polymer particles are analyzed in a rapid flow- through scanning device, wherein each particle is directly imaged using a fluorescence microscope and a camera.
  • Certain other embodiments are directed to a new "multiple-color genetic variation detection method" that can detect SNPs and kit using one chamber multiplexed endpoint PCR and differentially labeled allele-specific primers (one recognizing only the wild type allele and one only the mutant allele).
  • the primers can be differentially labeled with unique fluorophores (for example Cy3 and Cy5).
  • the primers are only extended by polymerase if the sequence at the 3 ' end of the primer shows perfect complementary to the target strand.
  • the differentially labeled wild type or mutant amplicons formed by extension of the respective labeled primers are each capable of binding to a probe that is complementary to a region of the allele immediately adjacent to the mutation site that is common to both the wild type and mutant alleles.
  • the color of the amplicon bound to the probes on the particle identifies the allele, mutant or wild type that was in the sample.
  • the sample comprising the plurality of nucleic acid targets
  • primer pairs for priming amplification of the plurality of nucleic acid targets, wherein the primer pairs hybridize to the targets at a primer annealing
  • the amount of that target amplified, detected and quantified will be zero.
  • the amount of nucleic acid in the target can be determined in step e) by comparing the quantified amplicon signal of step d) to the signal generated by amplification of an endogenous reference gene or a known amount of an external nucleic acid added into the sample, or by comparison to a standard curve plotting the signal generated by binding of the labeled nucleic acid target to a complementary probe over a broad range of target concentrations.
  • Labeled amplicons can be obtained using any method known in the art for PCR.
  • the labeling agent is bound to one primer of each of the primer pairs.
  • a preferred method uses direct hybridization of labeled primers onto target nucleic acids which generates labeled amplicons that hybridize to complementary probes immobilized on a particle.
  • a two probe system is used in which the labeling agent is bound to a free probe that is complementary to a region of the amplification product. The amplicon binds to less than all of the first probe sequence, leaving a portion of the amplicon unbound and free to bind to the second labeled probe.
  • either the sense or antisense strand of the target is labeled by labeling one or the other primer of each primer pair.
  • one of the primers of each pair is prelabeled with a fluorescent label such as 6-FAMTM, Alexa Fluor, Fluorescein, Phycoerythrin, Cy3, Cy5, Cy5.5, Dy 750, HEXTM, Iowa Black ®, IRDye ®, Joe, LightCyclerTM 640, MAX 550, Rhodamine GreenTM, Rhodamine RedTM, ROXTM, TETTM, TEX 615TM, Texas Red ®, TYETM 563, TYETM 665, TYETM 705, WellREDTM D2, WellREDTM D3, WellREDTM D4, TAMRA dyes such as those commercially from AnaSpecTM, TYE, or any other suitable fluorophore.
  • a fluorescent label such as 6-FAMTM, Alexa Fluor, Fluorescein, Phycoerythrin, Cy3, Cy5, Cy5.5,
  • indirect labeling of the amplicons is used, such as modifying one primer of each primer pair with a binding moiety such as a biotin group that will bind to a separate labeling agent such as fluorophore -modified streptavadin.
  • a binding moiety such as a biotin group that will bind to a separate labeling agent such as fluorophore -modified streptavadin.
  • Any substance that allows for detection of labeled amplicons may be used and there are many commercially available nucleic acid detection chemistries currently used in real-time PCR.
  • Labeling agents include molecular beacons, DNA binding (intercalating) dyes (such as ethidium bromide, proflavine, daunomycin, doxorubicin, and thalidomide), chromophores, quantum dots, radiolabels, carbon nanotubes, gold nanoparticles, Forster Resonance Energy Transfer (FRET) compounds, fluorophore quencher pairs, labels that interact by Forster Resonance Energy Transfer (FRET) including molecular beacons, binary nucleic acid probes, hydrolysis probes (also known as the TaqManTM assay (U.S. Pat. No. 5,210,015)), tagged primers, and
  • hybridization probes including two-probe systems.
  • labels and detection chemistries that are suitable for use in the present methods are described in, et al, US
  • PCR polymerase chain reaction
  • PCR requires a heat-stable DNA polymerase that preferably has a temperature optimum at around 70 °C, such as Taq polymerase.
  • a heat stable reverse transcriptase is also included in the chamber together with the DNA polymerase.
  • the PCR cocktail further includes: deoxynucleotide triphosphates (dNTPs); a buffer solution providing a suitable chemical environment for optimum activity and stability of the DNA polymerase; divalent cations, typically magnesium ions (Mg +); and monovalent cation potassium ions.
  • the sample containing the target nucleic acids is added to a chamber containing the DNA polymerase or reverse transcriptase and the PCR cocktail.
  • PCR cocktails are commercially available from various suppliers including New England Biosciences, Sigma- Aldrich, USB, Invitrogen, etc.
  • the present methods for one-chamber multiplex quantitative PCR with end point detection use thermal cycling to subject the PCR sample to a defined series of temperature steps. After an initial denaturation step, there are a series of amplification cycles, having a denaturation step, followed by an annealing step at a primer annealing temperature, followed by an extension step. Although annealing and extension are typically done at different temperatures, these two steps are optionally done at a common temperature. Typically, the primer annealing temperature is the same as or close to the primer melting temperature.
  • the primers should have as little secondary structure as possible and should be tested for hairpin formation and secondary structures.
  • compatible primer sets are designed that (1) have similar melting temperatures, (2) do not form hetero-dimers, and (3) are complementary to a region of the target nucleic acid (such as conserved regions of pathogen DNA that are unique for a particular species), and (4) are not complementary to the probe so that they do not prime the DNA probes immobilized on the particles.
  • the particles are hydrogel particles and the probes are dispersed throughout these porous particles.
  • Primers for use in the new methods typically range from about 10 to about 25 base pairs in length; however, the user may make longer or shorter primers.
  • primers and amplicons are preferably short to facilitate rapid hybridization kinetics.
  • primers for hydrogel particles are about 20 base pairs in length or shorter, and amplicons are preferably ⁇ 100 base pairs, preferably from about 50-70 base pairs in length.
  • primers may be attached to the particle so that the hybridization probes would not be required since the amplicons would be synthesized on the particles. Typically, only one primer of each primer pair would be attached to the particle. The other primer of the primer pair would be "free floating.”
  • DNA probes are designed for amplicon capture using any method known in the art.
  • probes typically incorporate about a 20 to 50 base pair region that is complementary to an interior region of the amplicon or the end region of the respective target nucleic acid that overlaps with the non- fluorescent primer target sequence.
  • the 3' end of the probes are phosphorylated or include an inverted dT or Dideoxycytidine (ddC) to block any potential unwanted PCR extension of the probes.
  • Customized DNA probes can be purchased from vendors, such as Integrated DNA Technologies, that are already modified with an acrydite group to facilitate direct incorporation of the probes into particles during hydrogel polymerization, as is described below.
  • oligonucleotides in a given buffer Common factors that increase the melting temperature are (1) a high degree of complementarity, (2) high salt concentrations, and (3) a high proportion of guanine and cytosine nucleotides. As temperature is increased near and beyond the Tm of a pair of oligonucleotides, hybridized complexes become less stable, and therefore de -hybridization will occur. This principle determines the specificity of hybridization, implying that hybridization should be carried out above the Tm of any undesirable oligonucleotide complexes but below that of the desired complexes to avoid loss of signal.
  • signal and specificity are optimized by selecting a hybridization temperature that is at least about 2 °C to about 15 °C higher than the selected primer annealing temperature, but lower than the Tm of target-probe complexes.
  • the primer annealing temperature is from about 35 °C to about 60 °C and the hybridization temperature is from about 37 to about 75 °C, This is accomplished by designing primers and probes that have melting temperatures relatively far apart to minimize unwanted amplicon/probe hybridization.
  • a primer is designed that has a T m of about 55°C
  • probes are designed that have a T m above about 70°C
  • primer annealing is conducted at a primer annealing temperature in the range of about 52-55°C
  • the hybridization step is conducted at a hybridization temperature of about 63-70°C, which is between the primer T m and the T m of the probe-amp licon complex.
  • probes can be modified with locked nucleic acids to increase the Tm of probe-amplicon complexes, even beyond the amplicon Tm. This is particularly attractive as hybridizations may be performed at temperatures above the temperature at which amplicons remain hybridized to their
  • hybridization times typically range from about 10 minutes to an hour or more; longer times are preferred to maximize sensitivity.
  • Samples of nucleic acid targets are prepared using standard procedures including biochemical purification, enrichment, immunological or physical treatments (PCR Technologies: Current Innovations, Thomas Weissensteiner, Hugh G. Griffin, Annette M. Griffin). There are also several commercial kits available for PCR sample preparation.
  • PCR Polymerase chain reaction
  • the particles are encoded hydrogel particles with more than one probe region permitting quantification of more than one target on a single particle and the amplicons are fluorescently labeled.
  • the signal from the labeled amplicons bound to the probes is detected and the amount of fluorescence emitted from each probe region on the particle is quantified, preferably by passing the particles one at a time through a flow cytometer equipped for fluorescence detection.
  • the amount of nucleic acid target in the complex sample is determined by comparing the quantified amplicon signal to either 1) a standard curve that correlates the signal generated by binding of a nucleic acid target to the corresponding probe over a broad range of target concentrations, 2) an endogenous reference gene or 3) by adding a known amount of an external target nucleic acid to the sample.
  • Methods for quantifying the amount of a nucleic acid target in a sample after PCR by comparison to an amplified endogenous reference gene such as beta-actin, GAPDH, nucleolar RNA or 18S ribosomal RNA are described in I. Nezarenko, et al, Nucleic Acids Research 2002, Vol. 30, No. 9 e37.
  • the results of the quantitative PCR using fluorogenic primers can be analyzed by the comparative C T method (User Bulletin 2, ABI PRISM 7700 Sequence Detection System, P/N 4303859). This method of analysis does not require plotting of a standard curve of C T versus starting copy number. Instead, the amount of target is calculated based on the difference between the C T of the target and an endogenous reference gene.
  • Example 3 describes quantifying the amount of target nucleic acids by comparison to an endogenous reference gene.
  • hydrogel particles are analyzed with high-throughput particle scanning in flow-focusing microfluidic devices that read the codes and determine the amount signal from the fluorescently labeled amplicons bound to the appropriate respective complementary probe region.
  • flow-focusing microfluidic devices that read the codes and determine the amount signal from the fluorescently labeled amplicons bound to the appropriate respective complementary probe region.
  • the devices are preferably scanned using slit illumination where the fluorescence intensity is measured using a photomultiplier module (such as Hamamatsu H7422TM) and particle code and target signal are decoded in real-time using custom written scripts.
  • a photomultiplier module such as Hamamatsu H7422TM
  • Example 1 multiplexed end point PCR.
  • Example 1 FIG. 1.
  • the results showed that encoded hydrogel particles provided single-nucleotide specificity with less than 3% cross-reactivity with sub- attomole sensitivity even without using probes with locked nucleic acids. This level of sensitivity far exceeds the -100 attomol sensitivity reported for the current state-of-the art bead- based system.
  • Probe sequences SEQ ID NOS: 3 and 4 were designed that (1) were complementary to a 40 base pair portion of the target sequence, (2) had a melting temperature T m above 72°C, and (3) were unable to form stable hybrids with the labeled primer due to a lack of complementarity.
  • the probe length of 40 base pairs was selected so that it formed a stable hybrid molecule with the amplicon at the 63 °C hybridization temperature.
  • Each probe was incorporated into the appropriate probe region of a barcoded hydrogel particle. Details of the experiment are set forth in Example 2.
  • a hybridization temperature of 63 °C that was 14 °C higher than the annealing temperature was used. As described above, the hybridization temperature is preferably 2-15 °C higher than the annealing temperature to maximize sensitivity and specificity.
  • the sample that now includes labeled amplicons #1 and #2 hybridized to respective probes #1 and #2 at known sites on the encoded hydrogel particles
  • hydrogel particles are sensitive to sub-attomole amounts of targeted nucleic acids as described above.
  • Hybridization times can vary. Because porous hydrogel particles have probe molecules dispersed throughout the matrix, a longer hybridization time of one hour were used to optimize diffusion of the labeled amplicons into the hydrogel. Hybridization times for hydrogel particles are preferably from about 10 minutes to about one hour. Other types of particles where probes are immobilized only on the outer surface typically require a shorter hybridization times.
  • Routine experimentation will determine the optimum hybridization time and hybridization temperature depending on the particles, primers and probes that are used.
  • the amount of fluorescence from each particle region was quantitated, and could be compared with a standard curve to quantitate the amount of the respective nucleic acid target in the original sample as is described below.
  • a qualitative positive result required probe- region fluorescence to be about 10X above the background noise.
  • the amount of target nucleic acid in the sample is meant both calculating the actual amount (the absolute number of copies of the target before amplification) and the relative amount of the target (normalized either to a known amount of exogenous DNA input or to endogenous reference genes/targets also called normalizing genes/targets).
  • the amount of a target amplified over multiple amplification cycles reaches a plateau after for example about 30 cycles and the amount is strongly affected by the amount of primers used.
  • the actual amounts of R A or DNA can be approximated by comparing the results to a standard curve produced by PCR using serial dilutions (eg. 1, 10, 100, 1000 copies) of a known amount of RNA or DNA.
  • the amount of a nucleic acid target in different samples is determined using a reference gene and dividing the measured amount of fluorescence from the labeled amplification products by the amount of fluorescence from a reference gene present in the same sample to normalize for possible variation between different samples.
  • a reference gene is selected that is expressed equally across all the samples.
  • HL60 human promyelocytic leukemia
  • DMSO fetal bovine serum
  • Upstream and downstream probe pairs designed for each of the targets by Peck were used to generate the amplicons.
  • Unique upstream biotin-conjugated primers for each target included a 24 nucleotide bar code sequence for each of the five mRNAs.
  • Applicants designed probes for immobilization on encoded hydrogel particles that were complementary to the 24 nucleotide bar code on each unique upstream primer to enable capture of the amplicons on encoded hydrogel particles. Methods for making the particles are described in Example 3.
  • Each encoded hydrogel particle was made with multiple probe regions directed to each of the five mRNAs (four targets and one reference gene) and a control, respectively.
  • the exact protocol described by Peck et al. was followed. There were 39 cycles in which: there was an initial denaturation at 92°C for 9 minutes, followed by denaturation at 92°C for 30 s, followed by annealing at 60°C for 30 s, extension at 72°C for 30 s for 39 cycles, and a final extension at 72°C for 5 minutes. After a final denaturation at 95°C for two minutes hybridization was carried out at 45°C for 60 minutes.
  • the hybridization temperature is from 2-15 °C higher than the primer annealing temperature to optimize specificity.
  • the hybridization temperature (45 °C) used by Peck et al. was 15 °C lower than the annealing temperature.
  • the respective Luminex and hydrogel particles were rinsed, incubated with streptavidin-phycoerythrin to fluorescently label the primers, rinsed again, and imaged for fluorescence.
  • the fluorescent signals obtained were quantified and normalized against the internal reference (LUA95) after subtracting the background signal from the control region on each particle (CTL).
  • the relative amount of fluorescence compared to the internal reference target after hybridization indicates the quantity of nucleic acid target (template) that was in the original sample. Note that for qualitative PCR, it is ideal to completely deplete the labeled primers to get the brightest signal possible. By contrast, for quantitative multiplexed end point PCR, it is not desirable to deplete the primers because the level of the signal is variable and depends on the number of target templates initially present in the sample. Table 1
  • Table 1 Raw and normalized data for a gene expression profiling experiment. For encoded hydrogel particles, signal calculated from five particles were averaged. The negative control signal [(-) CTL] was subtracted from each probe-region signal. These signals were then normalized using the internal reference (LUA95) to find the relative expression of each target in the samples. These expressions were compared for drug-treated and non-treated samples (by calculating the ratio). Differential expression is found by taking the logarithm of this expression ratio.
  • the new methods of the present invention are also suitable for detecting the presence of any pathogen, including DNA or RNA viruses, bacteria, and fungi, in a biological or environmental sample.
  • Example 3 provides a detailed outline for setting up and optimizing an assay for detecting and quantifying an influenza R A virus (such as influenza a (hlNl) and respiratory syncytial virus (RSV) (such as A-2) using commonly-targeted, highly-conserved genomic regions of the respective viruses as nucleic acid targets.
  • R A virus such as influenza a (hlNl) and respiratory syncytial virus (RSV) (such as A-2)
  • R A virus such as influenza a (hlNl)
  • RSV respiratory syncytial virus
  • FIG. 5 Persons of skill in the art can adapt these methods for setting up assays for other pathogens such as those causing sexually transmitted diseases (STDs).
  • STDs sexually transmitted diseases
  • multiple STDs such as N. gonorrhoeae and C.
  • trachomatis can be screened in a single specimen.
  • Another embodiment of the present invention is directed to a two-color method for detecting genetic variations including single nucleotide polymorphisms (SNPs) in a complex sample, which will enable the diagnosis and screening of many different genetic disorders. This will broadly benefit patients and carriers of undiagnosed genetic diseases and enabling high- throughput association studies for disorders not previously investigated due to cost prohibitive technology.
  • the present methods can be adapted as the genetic origins of more diseases are elucidated.
  • the genetic origins of these disorders may be monogenic as seen with disorders like cystic fibrosis, or may be extremely complex involving the interaction of several genes, as is the case with many cancers. More than 4,000 specific gene variants have been associated with common diseases such as heart disease, diabetes, asthma, and cancer. Rare diseases are those that occur at frequencies less than 1 in 2,000 people in a population, suggesting that these disorders affect only a small number of people. However, it is estimated that there are up to 8,000 rare diseases, cumulatively affecting up to 8% of the total population.
  • Genetic disorders can be caused by a number of DNA mutations including insertions, deletions, point mutations, single nucleotide polymorphisms (SNPs), and more. However, of the genetic variations observed in the human genome, nearly 90% are accounted for by SNPs.
  • hydrogel encoded particles provide sub- attomole sensitivity and single-nucleotide specificity with less than 3% cross-reactivity, even without the use of locked nucleic acids or altered probe design (FIG. 1), which makes them ideal for SNP detection.
  • Two methods for detecting single nucleotide mutations are known: (1) one uses allele-specific probes for selective target capture, and (2) one uses allele-specific primers for selective labeling of target genes.
  • An allele is one of a series of different forms of a gene, i.e. wild type and mutant forms.
  • One chamber multiplexed end point PCR can be used for the detection of genetic variation (including SNPs) using allele-specific probes that selectively hybridize to either the w ld type or one of the mutant alleles of a gene of interest.
  • Ordinary primers can be used with allele-specific probes as described above.
  • the allele specific probes have one or multiple locked nucleic acid (LNA) modifications in order to increase stability of properly hybridized alleles.
  • Probe Tm can cover a wide range, but certain
  • the probe Tm for SNP detection are near 55°C (or 57 - 59°C for LNA-modified probes)
  • For SNP detection need to keep probes short to get best discrimination, and they are capped, for example by 3 ' phosphorylation, to avoid incidental extension during PCR.
  • amplicons for SNP detection are from about 50 to about 70 base pairs in length, primers are preferably ⁇ 20 base pairs in length, and DNA probes are approximately 20 base pairs long (or shorter).
  • Separate probes are designed to target the interior region of each allele, such that the mutation site is approximately centered in the probe sequence.
  • the probe is 3 ' phosphorylated or otherwise capped, for example using inverted dT or ddC, to avoid incidental extension during PCR.
  • scanning is done using a single color and multiple probe regions.
  • one embodiment for SNP detection incorporates a single LNA-modified nucleotide at the SNP point mutation site on the allele-specific primers (for both the wild type or mutant primers) to raise the T m of a perfect primer/target match by 2-4 °C in order to optimize assay specificity. This creates a separation of melting temperatures for match and mismatched pairs making it easier to discriminate between the two.
  • the primers In the allele-specific primer amplification method, the primers would have the LNA. Only the appropriate allele will be amplified. In the other method using allele-specific probes, both alleles are amplified and an LNA is used on the probe to preferentially hybridize to only one of those.
  • Applicants also describe a method for detecting genetic variation (including SNPs) herein referred to as a "multiple-color genetic variation detection method," using one chamber multiplexed endpoint PCR and differentially labeled allele-specific primers.
  • An embodiment is directed to detection of genetic variation using different allele-specific primers that are differentially labeled with two or more distinct reporters such as two unique fluorophores (for example, Cy3 for a wild type allele and Cy5 for a mutant allele), and a single probe that can bind to either the wild type or mutant amplicon.
  • the primers could also be labeled with different radioisotopes or quantum dots, for example.
  • the probe is designed to be complementary to a region of the targeted gene that is adjacent to (flanks) or slightly overlapping the mutation site and therefore is common to both of the alleles being tested, for example a mutant and a wild-type allele. As such, each probe can bind either wild-type or mutant amplicons.
  • a comparison of the relative intensities of the two fluorophores determines which allele(s) is/are present in the sample.
  • a sample from a patient for example, would be screened to determine whether the patient carries the wild type allele or one of possibly many different mutant alleles. In other embodiments, up to six different alleles labeled respectively with six unique reporters can be used in this method.
  • Certain embodiments are directed to a method of amplifying and detecting genetic variation at a known mutation site in a gene in a sample comprising a plurality of nucleic acid targets by:
  • a first primer pair for priming amplification of a first allele of the gene using multiplex PCR wherein the primers hybridize to the targets at a primer annealing temperature, and wherein one primer of the first primer pair is labeled with a first reporter
  • a second primer pair for priming amplification of a second allele of the gene using multiplex PCR wherein the primers hybridize to the targets at a primer annealing temperature, and wherein one primer of the first primer pair is labeled with a second reporter
  • the present embodiments use allele-specific primers wherein the site of genetic variability is present at the 3' end of each allele-specific primer to assure accurate hybridization, i.e. the nucleotide residing at the mutation site in the gene of interest (either the wild type or mutant allele) is located at the 3' end of the primer. Unless there is 100 % complementarity to the target, the primer will not be extended. Thus, the primer for the mutant allele will only be extended if the targeted gene includes the particular mutation at the mutation site. If the patient has the mutant allele, the primer for the wild-type allele will not be able to form a stable hybrid with the gene.
  • primer design and assay conditions are optimized to ensure specific and accurate identification of all targets from heterozygous and homozygous alleles.
  • synthetic DNA can be used during optimization experiments in order to ensure that mutation and wild-type alleles are accurately discriminated.
  • cystic fibrosis the most common inherited autosomal recessive disorder, with a carrier frequency of 1 in 25 in the Caucasian population. This life threatening disease affects the respiratory, digestive, exocrine, and reproductive systems. While there are over 1000 known mutations that can lead to disease, the American College of Medical Genetics (ACMG) has recommended that 23 specific SNP mutations of the CF transmembrane regulator gene (CFTR) be including in CF screening Table 1. Watson, M.S., et al, Cystic fibrosis population carrier screening: 2004 revision of American College of Medical Genetics mutation panel. Genet Med, 2004. 6(5): p. 387-91.
  • ACMG American College of Medical Genetics
  • the detection of all 23 CFTR mutations with 100% specificity in a single sample is possible using the present one chamber, multiplexed dual color PCR assay for SNP detection.
  • an assay there would be allele-specific primers for each of the different SNPs with a primer for the corresponding wild type allele.
  • coded particles a single sample can be assayed for multiple SNPs.
  • the assay can be validated using genomic DNA from cystic fibrosis patients, carriers, and negative controls.
  • Genomic DNA can be obtained from patients, carriers, and negative which will provide both homo- and heterozygous alleles.
  • approximately 1,000 copies of DNA template could be used in 50 ⁇ PCR samples with about 30-40 amplification cycles for qualitative analysis using excess labeled primer.
  • Assay conditions can be optimized by persons of skill in the art depending on the particular experiment. It is not uncommon for PCR reactions to be nearly 100% efficient, meaning that product is doubled every single amplification cycle. Thus, for every molecule of starting template, there will be approximately 10 6 (1.7 attomol), 10 9 (1.8fmol), and 10 12 (1.8 pmol) product molecules after 20, 30, and 40 cycles, respectively. Serial dilutions of known target DNA or RNA ranging from 10 4 to ⁇ 1 can be used to determine the optimum amount of template for confident detection in a given assay. Run-to-run variability can be determined by repeating each test multiple times. Detection of ⁇ 10 copies of target DNA or RNA with an assay that requires less than 2 hours total is ideal and is possible with the new methods .
  • the concentration of fluorescent signal from the targets bound to the probes must be much greater than the background fluorescence from unreacted labeled primers in the solution.
  • the signal obtained from target capture is calculated to be sufficiently high to perform a 240-plex assay with signal-to-background ratio of 100, as found using the models developed in (Pregibon, et al, Anal. Chem. 2009, 81, 4873-4881) to estimate bound targets.
  • each PCR amplification cycle lasts only ⁇ 45 seconds - 5 minutes. This discrepancy in timescale further minimizes interference of the particles on PCR amplification.
  • the amount of free PCR primers diminishes over the amplification cycles by the time the hybridization step begins.
  • Hybridization is preferably accompanied by mixing to facilitate continuous sampling of the reaction mixture by the particles, though mixing is not required. In most cases, an excess of the primer that is labeled is used to ensure that there are target-strand amplicons free for hybridization.
  • this biasing is not necessary.
  • a typical assay about 30 particles per target nucleic acid are used (3 ⁇ 1 of particles at 10 particles/ ⁇ ) are pipetted into the chamber with them PCR product for a total sample volume of about 50 microliters in a 0.65 ml Eppendorf tube.
  • hybridization is carried out at a temperature of about 60 °C with an annealing temperature of about 55 °C for one hour with rapid mixing typically at 1800 rpm in a thermomixer (Rio, Quantafoil).
  • a one hour hybridization after the PCR amplification generated a signal obtained from target capture that was sufficiently high to perform a 240-plex assay with a signal to background ratio of about 100.
  • the hybridization times will vary depending on the particles used.
  • the present invention can also be used qualitatively.
  • the amount of primers used affects (1) the amount of product made, (2) the ratio of sense (detected) to anti-sense amplicons, and (3) the level of background fluorescence during scanning.
  • a model system to investigate the use of primers at varying concentrations and ratios can be used to optimize a particular assay to qualitatively access the presence of one or more nucleic acid targets in a sample.
  • Table 2 provides the design of a PCR primer concentration study with varying base concentrations and ratios for forward/reverse primers. In this study amplicons incorporating the forward primer labeled with Cy3 can be used subsequently for hybridization and detection. All concentrations should be orders of magnitude above the detection limit of the scanning system.
  • BSA or other molecules which act as blocking agents or detergents or surfactants may also improve the methods described herein.
  • BSA or other similar reagents known to those skilled in the art may improve a PCR reaction performed in a glass or quartz chamber by reducing the attraction of DNA and other reagents to the surfaces of the reaction chamber.
  • Other additives may be used which exhibit molecular crowding effects to improve the hybridization time, such as nucleic acid binding proteins or minor groove binders.
  • the poly(ethylene glycol) hydrogels a preferred embodiment for encoded particles, provide solution-like thermodynamics (i.e. strong binding) that allows high sensitivity and specificity, and three-dimensional probe distribution that gives a high target capacity to accommodate a wide dynamic range.
  • the particles are robust, withstanding high-temperatures used in PCR.
  • Hydrogels are a class of bio-friendly materials that characteristically retain water, allowing biological interactions to occur in three-dimensional space. Hydrogel materials (e.g. poly(ethylene glycol), PEG) are non-fouling, thus limiting non-specific interactions, and can derive from an extremely broad list of precursors. Nucleic acid hybridization in gels closely resembles that in solution, which enhances the sensitivity and specificity of nucleic acid detection. Fotin, A.V., et al., Parallel thermodynamic analysis of duplexes on
  • Hydrogel particles are synthesized using "flow lithography” that combines the non- mixing and continuous nature of micro f uidics with the precise sculpting of photolithography to control microparticle morphology, chemistry, and functionality.
  • flow lithography combines the non- mixing and continuous nature of micro f uidics with the precise sculpting of photolithography to control microparticle morphology, chemistry, and functionality.
  • Pregibon, D.C., M. Toner, and P.S. Doyle Multifunctional encoded particles for high-throughput biomolecule analysis.
  • Flow lithography is high- throughput (10 particles/hr), high resolution ( ⁇ 1 Dm), can form particles with multiple adjacent functionalities, and is applicable to any precursor material that reacts via free-radical
  • probe regions contain 20% PEG-diacrylate and 40% PEG, while encoded regions contain 30% of each.
  • the encoded region contains a fluorescent dye, such as rhodamine- methacrylate (Polysciences) for visualization.
  • the more rigid hydrogel composition includes about 30%> Poly(ethylene glycol) (700) diacrylate and about 30%>
  • DA30 Poly(ethylene glycol) (200), called DA30 hereinafter, where 700 and 200 refer to the molecular weights of the corresponding polymers; and the more porous hydrogel composition includes about 20% Polyethylene glycol) (700) diacrylate and about 40% Polyethylene glycol) (200), called DA20 hereinafter.
  • kits containing components for use in various embodiments of the multiplexed end point quantitative PCR assays Any of the components disclosed herein may be combined in a kit.
  • the kits include one or more primer pairs for priming amplification of one or more corresponding nucleic acid targets, preferably pre-labeled with fluorescent labels, and one or more particles on which are immobilized a plurality of probes complementary to a corresponding nucleic acid targets, preferably encoded hydrogel particles.
  • the particles are encoded polymer particles with multiple probe regions.
  • the kit may include probes that the user can complex with a desired particle. DNA polymerase and/or reverse transcriptase can also be included.
  • kits for detection of genetic variants such as SNP detection
  • the kit includes allele-specific pre-labeled primers as described above for -color detection using up to about six differentially labeled primers, wherein one primer is specific for a first allele such as a wild type allele, and the others are specific for other alleles showing the genetic variations, such as a mutant allele comprising the SNP.
  • the kit further includes probes that will bind to any of the allele-specific primers for the gene, which probes can be either free or more preferably immobilized onto particles, preferably hydrogel particles.
  • the kit can include a plurality of different particles each having a different probe or set of probes immobilized thereon, and a plurality of different primer pairs. If the primers are not pre-labeled, the kit optionally includes a detectable label that specifically binds to the primer.
  • Two probe kits come within the scope of the invention also, in which one probe is labeled or is modified to bind to a labeling agent.
  • kits may be packaged either in aqueous media or in lyophilized form.
  • the container means of the kits will generally include at least one container, into which a component may be placed, and preferably, suitably aliquoted. An appropriate number of containers will be included to accommodate each of the components in the kit. In some embodiments more than one component may be included in a single container.
  • the kits of the present invention also will typically include packaging for containing the various containers in close confinement for commercial sale.
  • the liquid solution may be an aqueous solution, with a sterile aqueous solution being particularly preferred.
  • certain components of the kit may be provided as dried powder(s).
  • the hydrogel particles may be dried.
  • the powder can be reconstituted by the addition of a suitable solvent that may be included in the kit or provided separately.
  • a kit may also include instructions for employing the kit components. Instructions may include variations that can be implemented.
  • hybridization means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases.
  • adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds.
  • Hybridization temperature as used herein specifically refers to the temperature at which amplicons hybridize to
  • “Complementary,” as used herein, refers to the capacity for precise pairing between two polynucleotides. For example, if a nucleotide at a certain position of a nucleic acid is capable of hydrogen bonding with a nucleotide at the same position of a DNA or RNA molecule, then the nucleic acid and the DNA or RNA are considered to be complementary to each other at that position. Perfect complementarity is not required throughout the length of the primers and probes. In the allele-specific primers, for example, the goal is to capture "similar" targets on the same probe, like the point mutated alleles that have been labeled with different fluorophores.
  • nucleic acids are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can hydrogen bond with each other.
  • “complementary” indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the two nucleic acids.
  • stringent conditions or “high stringency” are those conditions that allow hybridization between or within one or more nucleic acid strands containing complementary sequences, but preclude hybridization of non-complementary sequences. Such conditions are well known to those of ordinary skill in the art, and are preferred for applications requiring high selectivity.
  • Stringent conditions may comprise low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.15 M NaCl at temperatures of about 40 °C to about 70 °C for oligonucleotides of 20 - 50 base pairs. It is understood that the temperature and ionic strength of a desired stringency are determined in part by the length of the particular nucleic acids, the length and nucleobase content of the target sequences, the charge composition of the nucleic acids, and the presence or concentration of formamide, tetramethylammonium chloride or other solvents in a hybridization mixture.
  • Nucleic acids in the context of this invention include “oligonucleotides,” which refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or chimeras of both.
  • RNA ribonucleic acid
  • DNA deoxyribonucleic acid
  • oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally-occurring portions which function similarly.
  • backbone covalent internucleoside
  • Such modified or substituted oligonucleotides preferred over native forms to obtain enhanced affinity for the nucleic acid target and increased stability in the presence of nucleases.
  • a further preferred modification includes oligonucleotide primers or probes that include Locked Nucleic Acids (LNAs) in which the 2'-hydroxyl group is linked to the 3' or 4' carbon atom of the sugar ring thereby forming a bicyclic sugar moiety.
  • the linkage is preferably a methelyne ( ⁇ CH 2 ⁇ ) n group bridging the 2' oxygen atom and the 4' carbon atom wherein n is 1 or 2.
  • LNAs and preparation thereof are described in WO 98/39352 and WO 99/14226.
  • Other modifications include 2'-methoxy(2' ⁇ 0 ⁇ CH 3 ), 2'-aminopropoxy (2' ⁇
  • the 2'-modification may be in the arabino (up) position or ribo (down) position.
  • a preferred 2'-arabino modification is 2'-F.
  • Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide.
  • Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.
  • Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos.: 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;
  • Oligonucleotides may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions.
  • nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine. (C) and uracil (U).
  • Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2- propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2- thiocytosine, 5-halouracil and cytosine, and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8- thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5- bromo, 5-trifluoromethyl and other 5 -
  • nucleobases include tricyclic pyrimidines such as phenoxazine cytidine(lH-pyrimido[5,4-b][l,4]benzoxazin- 2(3H)-one), phenothiazine cytidine (lH-pyrimido[5,4-b][l,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g.
  • nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.
  • Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al, Angewandte Chemie,
  • nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention.
  • These include 5 -substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5- propynyluracil and 5-propynylcytosine. 5 -methyl cytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C. (Sanghvi, Y. S., Crooke, S. T.
  • the present invention also includes self-contained nucleotide -based bioanalytical systems incorporated into portable devices that incorporate the polymer particles or other particles for nucleotide capture, detection, and quantitation using the methods of the present invention.
  • Advances in system miniaturization make it possible to combine sample preparation, amplification and detection in a single portable device that may include cartridges containing chambers, channels, and/or heating or cooling elements to store and manipulate samples and reagents and/or scan particles as described for various embodiments of the multiplexed end point quantitative PCR assays. Any of the components and procedures disclosed herein may be combined with cartridge-based analysis.
  • the cartridges include chambers containing one or more primer pairs for priming amplification of one or more corresponding nucleic acid targets, preferably pre-labeled with fluorescent labels, and one or more particles on which are immobilized a plurality of probes complementary to a corresponding nucleic acid targets, preferably encoded hydrogel particles.
  • the cartridges may contain heating or cooling elements to perform amplification, as well as flow-focusing channels to align and flow particles for analysis.
  • the cartridges are to be interfaced with equipment bearing active elements to deliver samples, manipulate flows, provide direct or indirect heating and cooling, illuminate the sample, and acquire fluorescent or other emitted signal.
  • Cartridges may be disposable or reusable.
  • Hand-held or portable devices can be used in point-of-care facilities such as doctor's offices or hospitals, veterinarian's offices, pharmacies, diagnostics labs, and clinics; and also for detecting biological threats in civilian or military areas.
  • point-of-care facilities such as doctor's offices or hospitals, veterinarian's offices, pharmacies, diagnostics labs, and clinics; and also for detecting biological threats in civilian or military areas.
  • a review of portable nucleic acid bioanalytical systems is provided by T. M. Lee et al., DNA-based bioanalytical microsystems for handheld device applications, Analytica Chemica, Acta 556 (2006) 26-37; see also T.Ray, UK Startup DNA Electronics Developing Handheld Device to Detect Genetic Risk for Drug AEs, Pharmaco genomics Reporter - February 25, 2009; and K. P.
  • the devices are suitable also for use in use in non- healthcare industries like food preparation, agriculture, and animal farming.
  • the devices are preferably small in size ( ⁇ 8" in all dimensions) and perform the assays described herein in less than two hours from the moment a sample is introduced.
  • the devices contain active elements such as pumps, valves, optics, detectors, and electronics to introduce, manipulate, and interrogate samples. In certain embodiments, the devices may be used with cartridges or kits.
  • the devices may contain reservoirs containing specific reagents including prelabeled primers, particles on which are immobilized one or more probes that detect amplicons of the targeted nucleic acids, PCR reagents, etc. as described herein.
  • the devices may provide the capability to analyze one or multiple samples simultaneously.
  • Example 1 Encoded hydrogel particle sensitivity, specificity and thermal stability
  • Particles bearing four unique probe regions were synthesized, each probe region containing a unique probe for each of the four let-7 family members (7a - 7d), which vary by only one or two nucleotides in sequence.
  • the probes were incorporated at a precursor concentration of 10 ⁇ .
  • Particles were incubated with samples containing 5 femtomoles of biotinylated let-7a RNA and 500 ng of total E. coli RNA to add complexity, thus mimicking a "real" assay that would likely involve total human RNA consisting of broadly heterogeneous nucleic acid mixtures. Incubations were one hour at 58°C with 0.5M NaCl in the hybridization buffer.
  • Encoded hydrogel particles need to be stable upon heating to withstand the high- temperature demands of PCR reaction.
  • the results of experiments shown in (FIG. 2 ) verify that target hybridization signal is identical with and without extensive heating.
  • This experiment also demonstrates the sensitive/specific quantification of fluorescent target without post-hybridization rinsing, which is important for a "one-pot" assay.
  • Particles bearing two probe regions, a negative control region, and barcode region were heated at 95 °C for 20 minutes. They were then incubated with 500 attomole target at 37°C for 60 minutes and imaged immediately afterward. Particles subjected to heating gave similar fluorescent signal as those which were not heated. No rinsing steps were required for this assay.
  • the stability of the hydrogel particles at 95°C for 20 minutes was not a foregone conclusion since the particles have relatively high water content and could have either deformed so that they could not be readily scanned, or sensitivity could have been reduced.
  • particles can be made with about 3,000 different unique identification codes.
  • Multiplex encoded polymer particles were synthesized using flow lithography, and are described at length in US. Serial No. US2007/0105972, and US 2008/0176216, Pregibon, D.C., M. Toner, and P.S. Doyle, Multifunctional encoded particles for high-throughput biomolecule analysis. Science, 2007. 315(5817): p. 1393-1396, Pregibon, D.C. and P.S. Doyle, Optimization of Encoded Hydrogel Particles for Nucleic Acid Analytical Chemistry, Vol. 81, No. 12, June 15, 2009, 4873-4881, incorporated herein by reference.
  • ⁇ -phage DNA target sequences were selected that were 60 base pairs long and primers were selected that had no significant homology to homo sapiens in a BLAST search.
  • the sense strand of the ⁇ -phage DNA was amplified, therefore the reverse primer was designed to incorporate a 5' Cy3 modification for fluorescent detection of the amplicons.
  • a single type of hydrogel particle was designed for this study, with rhodamine- methacrylate (Polysciences) for visualization of the encoded region, an inert negative control region flanking the encoded region, that in turn was flanked by two probe regions: one probe region containing probe #1 specific for amplicon #1, and the second probe region at the end of the particle with probe #2 specific for amplicon #2.
  • rhodamine- methacrylate Polysciences
  • PCR amplification and hybridization was done in a single tube in a ⁇ volume using: Taq polymerase (IX, diluted from 5X master mix - New England BiolabsTM),
  • the initial PCR denaturation was 5 minutes at 95°C followed by 30 cycles of amplification that included a 30 second 95°C denaturation, 30 second 49°C primer annealing, and 45 second 72°C extension. A final extension of 5 minutes at 72°C occurred prior to a 1 minute 95°C denaturation. Finally the sample was cooled to the probe hybridization temperature of 63°C to promote stringent hybridization. Hybridization was promoted by shaking at 1800 rpm while maintaining a temperature of 63°C for one hour using a thermomixerTM (Rio, Quantafoil).
  • a primer annealing temperature of 49°C was used in these experiments for the primer with a melting temperature of 55°C
  • a higher primer annealing temperatures could have been used, for example up to about 55°C.
  • the user can vary the temperatures using routine experimentation to optimize specificity.
  • HL60 human promyelocytic leukemia
  • HL60 human promyelocytic leukemia
  • RPMI fetal bovine serum
  • DMSO dimethyl sulfoxide
  • Total RNA was isolated from bulk cultures with TRIzol Reagent (Invitrogen, Carlsbad, CA, USA), in accordance with the manufacturer's directions.
  • microtiter plate cultures were treated with 200 nmol/1 tretinoin or DMSO for two days to mimic the submaximal signatures likely to be encountered in a small molecule screen, and were and prepared for mRNA capture by the addition of Lysis Buffer (RNAture, Irvine, CA, USA).
  • upstream probes were composed (5' to 3') of the complement of the T7 primer site (SEQ ID NO: 7: TAA TAC GAC TCA CTA TAG GG), a 24 nucleotide (nt) barcode, and a 20 nucleotide gene-specific sequence.
  • Downstream probes were 5'-phosphorylated, and contained a 20 nucleotide gene-specific sequence and the T3 primer site (SEQ ID NO: 8: TCC CTT TAG TGA GGG TTA AT). Barcode sequences were developed by Tm Bioscience (Toronto, Ontarion, Canada) and detailed in the Luminex FlexMAP Microspheres Product Information Sheet.
  • Probe sequences are provided in Additional data file 2 of [Peck et al, Genome Biology, 7:R61, 2006]. Capture probes contained the complement of the barcode sequences and had 5 '-amino modification and a C12 linker.
  • the T7 primer (SEQ ID NO: 7: 5 * -TAA TAC GAC TCA CTA TAG GG- 3 * ) was 5'- biotinylated.
  • the T3 primer has SEQ ID NO: 9: 5 * - ATT AAC CCT CAC TAA AGG GA-3 * .
  • Oligonucleotides (all with standard desalting) were from Integrated DNA Technologies
  • Luminex xMAP Multi-Analyte COOH Microspheres were coupled to capture probes in a semi- automated microtiter plate format. Approximately 2.5 x 106 microspheres were dispensed to the wells of a V-bottomed microtiter plate, pelleted by centrifugation at 1800 g for 3 minutes, and the supernatant removed. Beads were resuspended in 25 ⁇ binding buffer (0.1 M 2- [N- morpholino]ethansulfonic acid; pH 4.5) by sonication and pipeting, and 100 pmol capture probe was added.
  • 25 ⁇ binding buffer 0.1 M 2- [N- morpholino]ethansulfonic acid; pH 4.5
  • Coupled microspheres were resuspended in 50 ⁇ TE (pH 8.0) and stored in the dark at 4°C for up to one month.
  • Bead mixes were freshly prepared and contained about 1.5 x 105/ml of each microsphere in 1.5 X TMAC buffer (4.5 mol/1 tetramethylammonium chloride, 0.15% N-lauryl sarcosine, 75 mmol/1 tris-HCl [pH 8.0], and 6 mmol/1 EDTA [pH 8.0]).
  • 1.5 X TMAC buffer 4.5 mol/1 tetramethylammonium chloride, 0.15% N-lauryl sarcosine, 75 mmol/1 tris-HCl [pH 8.0], and 6 mmol/1 EDTA [pH 8.0].
  • the mapping of bead number to capture probe sequence is provided in Additional data file 3 of [Peck et al, Genome Biology, 7:R61, 2006].
  • transcripts were captured in oligo-dT coated 384 well plates (GenePlateHT; RNAture) from total RNA (500 ng) in Lysis Buffer (RNAture) or whole cell lysates (20 ⁇ ). Plates were covered and centrifuged at 500 g for one minute, and incubated at room temperature for one hour. Unbound material was removed by inverting the plate onto an absorbent towel and spinning as before. A volume of 5 ⁇ of an M-MLV reverse transcriptase reaction mix (Promega, Madison, WI, USA) containing 125 ⁇ / ⁇ of each dNTP (Invitrogen) was added.
  • GenePlateHT RNAture
  • M-MLV reverse transcriptase reaction mix Promega, Madison, WI, USA
  • the plate was covered, spun as before, and incubated at 37°C for 90 minutes. Wells were emptied by centrifugation, as before. A volume of 10 frnol of each probe was added in ⁇ *Taq Ligase Buffer (New England Biolabs, Ipswich, Ma, USA; 5 ⁇ ), the plate covered, spun as before, heated at 95°C for two minutes and maintained at 50°C for six hours. Unannealed probes were removed by centrifugation, as before. A volume of 5 ⁇ of l x Taq Ligase Buffer containing 2.5 U Taq DNA ligase (New England Biolabs) was added, the plate covered, spun as before, and incubated at 45°C for one hour followed by 65°C for 10 minutes.
  • ⁇ *Taq Ligase Buffer New England Biolabs, Ipswich, Ma, USA; 5 ⁇
  • MFIs median fluorescence intensities
  • Total time from the start of hybridization to download of raw data from the instrument for 96 samples processed in parallel in a single microtiter plate is approximately three hours.
  • Expression values for each transcript were corrected for background signal by subtracting the MFI of corresponding bead sets from blank (TE only) wells. Values below an arbitrary baseline (5) were set to 5, and all were normalized against an internal control feature (GAPDH3).
  • PEG- DA, Mi 700
  • PEG Mw -200, -50 cP at 25 °C, Aldrich
  • Particles were rinsed two times in phosphate buffered saline containing 0.05% Tween-20 by adding 500ul of rinse buffer to the tubes, centrifuging for 30seconds, and aspirating ⁇ 500ul from the tube.
  • NIH Image was used to visualize images captured from an EB-CCD camera (C7190-20, Hamamatsu) mounted to the side port of the microscope with camera settings of 6.6, 1.3, and 2.9 for gain, offset, and sensitivity, respectively.
  • Example 4 Example 4
  • Detection of respiratory diseases such as influenza a (hlNl) and RSV (such as A-2)
  • the one chamber multiplexed quantitative end point PCR assay can be adapted for amplifying and detecting RNA or DNA viruses, exemplified here using Human Influenza A (HlNl) and Respiratory Syncytial Virus (RSV) using the one chamber multiplexed quantitative end point PCR by following the basic steps below.
  • HlNl Human Influenza A
  • RSV Respiratory Syncytial Virus
  • Primer sets will be designed to make amplicons of pathogen-specific nucleic acid targets, each ⁇ 100 base pairs in length modified with a single Cy3 fluorophore. Primer concentration, PCR cycles, and hybridization time on detection signal will be varied to optimize the assay ideally to achieve reproducible detection of ⁇ 10 RNA templates per target nucleic acid. For RNA viruses, it is necessary to first produce cDNA libraries prior to PCR amplification. Stockton, J., et al., Multiplex PCR for typing and subtyping influenza and respiratory syncytial viruses. J Clin Microbiol, 1998. 36(10): p. 2990-5.
  • primers sets as described herein will be identified for amplifying targeted pathogen nucleic acid using a primer-design program (such as Invitrogen's OligoPrimerTM for instance) to search through a user-input genomic sequence, identifying sets of primers that provide the desired primer melting temperatures and amplicon sizes. Ideal target regions of pathogen RNA are unique for the species and highly conserved. Each potential primer identified will be assessed for species-specificity via BLAST search. A script written in MATLAB or freely available software (like UNAfold), for example, can be used to assess dimer-formation with all other primers. Primers are preferably designed to have melting temperatures near 55°C, be ⁇ 20 base pairs in length, and provide amplicons ⁇ 60 base pairs in size. The primer incorporated into the amplicon is ideally prelabeled for example with a single Cy3 label for fluorescence detection,
  • Testing can be done using incubations with -30 particles (3 ⁇ 1 of particles at 10 particles/ ⁇ ) pipetted into mPCR product for a total sample volume of 52 ⁇ in 0.65 ml
  • Phage MS2 RNA Once the basic parameters are established with Phage MS2 RNA, the assay will be run to amplify species-specific targets from Phage MS2 RNA, Infiuenza A and RSV genomic RNA, and use hybridization assays with all three particles types in a single sample.
  • Amplification via PCR is an exponential process. As such, in an assay the actual number of templates in the PCR reaction is not being quantified, instead the number captured on hydrogel particles is determined. By knowing the amplification efficiency of a given target along with the number of amplification cycles, the amount of amplicon captured on a particle can then be used to estimate the nucleic acid templates initially present. Using the optimized primer concentrations, the optimum number of PCR cycles for a known amount of target can be determined.
  • Hybridization time and temperature can be varied to find optimum conditions, starting for example using a hydrogel particle with a probe having a melting temperature of about 70 degrees and primers having a melting temperature of about 55 degrees. To calibrate, each test is repeated 10 times to assess reproducibility and specificity at varying pathogen concentrations. Using methods known in the art, (1) sensitivity, (2) specificity, (3) inter-run variance, and (4) run-to-run variance of the assays are determined and adjusted to optimum conditions. [0125] Once the assay is optimized, clinically-relevant samples, including throat swabs and sputum specimens, will be analyzed.
  • PCR inhibitors e.g. melanin
  • tests are run in the presence of inhibitors to choose conditions to assure that the Multiplexed End-Point PCR assays reproducibly detect inhibition, including optimizing the concentration of the positive control to accurately reflect the presence of an inhibitor.

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Abstract

Dans certains modes de réalisation, l'invention se rapporte à des procédés de PCR quantitative multiplexée en récipient unique destinés à l'analyse en point final d'une pluralité d'acides nucléiques cibles dans un échantillon complexe sans intervention de l'utilisateur, et à diverses particules codées sur lesquelles sont immobilisées une ou plusieurs sondes qui s'hybrident avec la pluralité de cibles. Dans d'autres modes de réalisation, l'invention porte sur un nouveau « procédé de détection de multiples variations génétiques de couleur » qui permet de détecter des SNP, et sur une trousse faisant appel à une PCR en point final multiplexée en récipient unique et à des amorces spécifiques d'allèle étiquetées de façon différentielle (l'une ne reconnaissant que l'allèle de type sauvage et l'autre ne reconnaissant que l'allèle mutant).
PCT/US2009/066778 2009-12-04 2009-12-04 Analyse en point final d'une pcr quantitative multiplexée d'acides nucléiques cibles Ceased WO2011068518A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
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WO2013074874A1 (fr) * 2011-11-17 2013-05-23 Clarient Diagnostic Services, Inc. Procédé d'amplification spécifique d'un allèle
US10519490B2 (en) 2016-05-03 2019-12-31 Korea Institute Of Science And Technology Porous matrix comprising nucleic acid primer-carbon material composites and PCR using the same
US10648024B2 (en) 2013-10-28 2020-05-12 Korea Institute Of Science And Technology Porous structure and method for manufacturing same
CN112575118A (zh) * 2020-07-15 2021-03-30 江苏硕世生物科技股份有限公司 一种利用熔解曲线同时检测多种腹泻病毒的方法
CN113272447A (zh) * 2018-11-08 2021-08-17 脱其泰有限责任公司 用于液滴中颗粒多重处理的系统和方法

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US20030143604A1 (en) * 2001-11-30 2003-07-31 Storhoff James J. Real-time monitoring of PCR amplification using nanoparticle probes
US20050147973A1 (en) * 2002-03-26 2005-07-07 Tim Knott Immobilized probes
US20060147924A1 (en) * 2002-09-11 2006-07-06 Ramsing Neils B Population of nucleic acids including a subpopulation of lna oligomers
US20060228742A1 (en) * 2003-05-15 2006-10-12 Ghazala Hashmi Correcting an assay image of an array of signals generated from a multiplexed hybridization-mediated assay
US20090036316A1 (en) * 2003-02-26 2009-02-05 Complete Genomics, Inc. Random array DNA analysis by hybridization

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030143604A1 (en) * 2001-11-30 2003-07-31 Storhoff James J. Real-time monitoring of PCR amplification using nanoparticle probes
US20050147973A1 (en) * 2002-03-26 2005-07-07 Tim Knott Immobilized probes
US20060147924A1 (en) * 2002-09-11 2006-07-06 Ramsing Neils B Population of nucleic acids including a subpopulation of lna oligomers
US20090036316A1 (en) * 2003-02-26 2009-02-05 Complete Genomics, Inc. Random array DNA analysis by hybridization
US20060228742A1 (en) * 2003-05-15 2006-10-12 Ghazala Hashmi Correcting an assay image of an array of signals generated from a multiplexed hybridization-mediated assay

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013074874A1 (fr) * 2011-11-17 2013-05-23 Clarient Diagnostic Services, Inc. Procédé d'amplification spécifique d'un allèle
US10648024B2 (en) 2013-10-28 2020-05-12 Korea Institute Of Science And Technology Porous structure and method for manufacturing same
US10519490B2 (en) 2016-05-03 2019-12-31 Korea Institute Of Science And Technology Porous matrix comprising nucleic acid primer-carbon material composites and PCR using the same
CN113272447A (zh) * 2018-11-08 2021-08-17 脱其泰有限责任公司 用于液滴中颗粒多重处理的系统和方法
CN112575118A (zh) * 2020-07-15 2021-03-30 江苏硕世生物科技股份有限公司 一种利用熔解曲线同时检测多种腹泻病毒的方法
CN112575118B (zh) * 2020-07-15 2023-12-01 江苏硕世生物科技股份有限公司 一种利用熔解曲线同时检测多种腹泻病毒的方法

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