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WO2005065290A2 - Metallisation d'acides nucleiques par sols metalliques, et utilisations correspondantes - Google Patents

Metallisation d'acides nucleiques par sols metalliques, et utilisations correspondantes Download PDF

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Publication number
WO2005065290A2
WO2005065290A2 PCT/US2004/043530 US2004043530W WO2005065290A2 WO 2005065290 A2 WO2005065290 A2 WO 2005065290A2 US 2004043530 W US2004043530 W US 2004043530W WO 2005065290 A2 WO2005065290 A2 WO 2005065290A2
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Prior art keywords
nucleic acid
metal
acid molecule
probes
sample
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WO2005065290A3 (fr
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Dennis M. Connolly
Charles Deboer
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Integrated Nano Technologies LLC
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Integrated Nano Technologies LLC
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical

Definitions

  • This invention relates to methods for metallizing nucleic acid molecules, and uses thereof.
  • Nucleic acids such as DNA or RNA
  • Powerful new molecular biology technologies enable one to detect congenital or infectious diseases. These same technologies can characterize DNA for use in settling factual issues in legal proceedings, such as paternity suits and criminal prosecutions.
  • amplification of a small amount of nucleic acid molecules isolation of the amplified nucleic acid fragments, and other procedures are necessary.
  • the science of amplifying small amounts of DNA have progressed rapidly and several methods now exist. These include linked linear amplification, ligation-based amplification, transcription-based amplification and linear isothermal amplification.
  • Ligation-based amplification includes the ligation amplification reaction (LAR) described in detail in Wu et al., Genomics 4:560 (1989) and the ligase chain reaction described in European Patent No. 0320308B 1.
  • Transcription-based amplification methods are described in detail in U.S. Patent Nos. 5,766,849 and 5,654,142, Kwoh et al., Proc. Natl. Acad. Sci. U.S.A. 86:1173 (1989), and PCT Publication No. WO 88/10315 to Ginergeras et al.
  • PCR reaction is based on multiple cycles of hybridization and nucleic acid synthesis and denaturation in which an extremely small number of nucleic acid molecules or fragments can be multiplied by several orders of magnitude to provide detectable amounts of material.
  • One of ordinary skill in the art knows that the effectiveness and reproducibility of PCR amplification is dependent, in part, on the purity and amount of the DNA template.
  • Certain molecules present in biological sources of nucleic acids are known to stop or inhibit PCR amplification (Belec et al., Muscle and Nerve 21(8): 1064 (1998); Wiedbrauk et al., Journal of Clinical Microbiology 33(10):2643-6 (1995); Deneer and Knight, Clinical Chemistry 40(l):171-2 (1994)).
  • hemoglobin, lactoferrin, and immunoglobulin G are known to interfere with several DNA polymerases used to perform PCR reactions (Al-Soud and Radstrom, Journal of Clinical Microbiology 39(2):485-493 (2001); Al-Soud et al., Journal of Clinical Microbiology 38(l):345-50 (2000)).
  • These inhibitory effects can be more or less overcome by the addition of certain protein agents, but these agents must be added in addition to the multiple components already used to perform the PCR.
  • the removal or inactivation of such inhibitors is an important factor in amplifying DNA from select samples.
  • nucleic acid sequencer and fragment analyzer in which gel electrophoresis and fluorescence detection are combined.
  • electrophoresis becomes very labor-intensive as the number of samples or test items increases.
  • VLSIPSTM New technology
  • New technology has enabled the production of chips smaller than a thumbnail where each chip contains hundreds of thousands or more different molecular probes.
  • These techniques are described in U.S. Patent No. 5,143,854 to Pirrung et al., PCT Publication No. WO 92/10092, and PCT WO 90/15070.
  • These biological chips have molecular probes arranged in arrays where each probe ensemble is assigned a specific location. These molecular array chips have been produced in which each probe location has a center to center distance measured on the micron scale.
  • array chips have been useful in a number of different types of scientific applications, including measuring gene expression levels, identification of single nucleotide polymorphisms, and molecular diagnostics and sequencing as described in U.S. Patent No. 5,143,854 to Pirrung et al. [0008] Array chips where the probes are nucleic acid molecules have been increasingly useful for detection of the presence of specific DNA sequences. Most technologies related to array chips involve the coupling of a probe of known sequence to a substrate that can either be structural or conductive in nature.
  • Structural types of array chips usually involve providing a platform where probe molecules can be constructed base by base or by covalently binding a completed molecule.
  • Typical array chips involve amplification of the target nucleic acid followed by detection with a fluorescent label to determine whether target nucleic acid molecules hybridize with any of the oligonucleotide probes on the chip. After exposing the array to a sample containing target nucleic acid molecules under selected test conditions, scanning devices can examine each location in the array and quantitate the amount of hybridized material at that location.
  • conductive types of array chips contain probe sequences linked to conductive materials such as metals. Hybridization of a target nucleic acid typically elicits an electrical signal that is carried to the conductive electrode and then analyzed.
  • PNA protein nucleic acid
  • LNA locked nucleic acid
  • methyl phosphonate chemistries PNA (protein nucleic acid)
  • PNA protein nucleic acid
  • LNA locked nucleic acid
  • methyl phosphonate chemistries PNA (protein nucleic acid)
  • all of the DNA analogs have higher melting temperatures than standard DNA oligonucleotides and can more easily distinguish between a fully complementary and single base mis-match target. This is possible because the DNA analogs do not have a negatively charged backbone, as is the case with standard DNA. This allows for the incoming strand of target DNA to bind tighter to the DNA analog because only one strand is negatively charged.
  • the most studied of these analogs for hybridization techniques is the PNA analog, which is composed of a protein backbone with substituted nucleobases for the amino acid side chains (see www.appliedbiosystems.com or www.eurogentec.com).
  • PNAs have been used in place of standard DNA for almost all molecular biology techniques including DNA sequencing (Arlinghaus et al., Anal Chem. 69:3747-53 (1997)), DNA fingerprinting (Guerasimova et al., Biotechniques 31 :490-495 (2001 )), diagnostic biochips (Prix et al., Clin. Chem. 48:428-35 (2002); Feriotto et al., Lab Invest 81:1415-1427 (2001)), and hybridization based microarray analysis (Weiler et al., Nucleic Acids Res 25:2792-2799 (1997); Igloi, Genomics 74:402-407 (2001)).
  • sequences on a substrate are known.
  • the sequences may be formed according to the techniques disclosed in U.S. Patent No. 5,143,854 to Pirrung et al., PCT Publication No. WO 92/10092, or U.S. Patent No. 5,571,639 to Hubbell et al.
  • a metal electrical conductor has no reactive sites, in principle, except those that may be adventitiously or deliberately positioned on the surface of the metal.
  • Hybridization of target DNAs to such surface bound capture probes poses difficulties not seen, if both species are soluble. Steric effects result from the solid support itself and from too high of a probe density. Studies have shown that hybridization efficiency can be altered by the insertion of a linker moiety that raises the complementary region of the probe away from the surface (Schepinov et al., Nucleic Acid Res. 25:1155-1161 (1997); Day et al., Biochem J. 278:735-740 (1991)), the density at which probes are deposited (Peterson et al., Nucleic Acids Res. 29:5163- 5168 (2001); Wilkins et al., Nucleic Acids Res.
  • the present invention is directed to achieving these objectives.
  • One aspect of the present invention relates to a method for metallizing one or more sites of a nucleic acid molecule.
  • This method involves providing a metal sol and contacting the metal sol and a nucleic acid molecule under conditions effective to mordant metal particles from the metal sol on one or more sites of the nucleic acid molecule.
  • the mordanted nucleic acid molecule is then contacted with a metal deposition solution under conditions effective to deposit metal from the metal deposition solution on the nucleic acid molecule.
  • Another aspect of the present invention relates to a method for detecting a target nucleic acid molecule in a sample.
  • This method involves providing a device for detecting the presence of a target nucleic acid molecule in a sample.
  • the device has two electrical conductors, including a first and second electrical conductor, which are not in contact with one another.
  • the device also has one or more sets of two oligonucleotide probes attached to the electrical conductors.
  • the probes are positioned such that they cannot come into contact with one another and such that a target nucleic acid molecule, which has two sequences, a first sequence complementary to a first probe attached to the first electrical conductor and a second sequence complementary to a second probe attached to the second electrical conductor, can bind to both probes.
  • the probes are contacted with a sample which may have the target nucleic acid molecule under selective hybridization conditions to permit target nucleic acid molecules, if any, present in the sample to hybridize to both of the probes.
  • a metal sol is provided. The metal sol and the device are contacted after the probes are contacted with the sample under conditions effective to mordant metal particles from the metal sol on any of the target nucleic acid molecules hybridized to the probes.
  • the mordanted target nucleic acid molecule is contacted with a metal deposition solution under conditions effective to deposit metal from the metal deposition solution on to any of the target nucleic acid molecules hybridized to the probes. It is then determined if an electrical current can be carried between the probes, the electrical current between the probes indicating the presence of the target nucleic acid molecule in the sample.
  • Figures 1 A-B show a perspective view of a system for detection of a target nucleic acid molecule from a sample which includes a desk-top detection unit and a cartridge which is inserted into the desk-top unit.
  • Figure 1C shows a schematic view of this system.
  • Figure 2 A depicts a single test structure on a detection chip suitable to be positioned in first chamber 20 of the system shown in Figures 1 A-C, where oligonucleotide probes are attached to electrical conductors in the form of spaced apart conductive fingers.
  • Figure 2B shows how a target nucleic acid molecule present in a sample is detected by the detection chip.
  • Figures 3 A-B show a perspective view of a system for detection of a target nucleic acid molecule which includes a portable detection unit and a cartridge which is inserted into the portable unit.
  • Figure 3C shows a schematic view of this system.
  • the present invention relates to a method for metallizing one or more sites of a nucleic acid molecule.
  • This method involves providing a metal sol and contacting the metal sol and a nucleic acid molecule under conditions effective to mordant metal particles from the metal sol on one or more sites of the nucleic acid molecule.
  • the mordanted nucleic acid molecule is then contacted with a metal deposition solution under conditions effective to deposit metal from the metal deposition solution on the nucleic acid molecule.
  • metal from a metal sol is mordanted onto one or more sites of a nucleic acid molecule.
  • Mordanting is a process well known to those skilled in the art of photographic printing, and in the art of dying cloth. It is known in the photographic art to use various polymeric materials and mordants in color image transfer elements comprising a support and layer containing a silver halide emulsion to prevent the migration of dyes. Receiving elements containing mordants are described for example in U.S. Patent Nos. 2,584,080 and 3,770,439, each of which is hereby incorporated by reference in its entirety.
  • mordanting Although the exact mechanism of mordanting is often unclear, the process is one wherein a soluble molecule, upon encountering a mordanting species, forms a fixed complex with the mordant, and is no longer able to move away from the mordant, even when repeatedly washed with water or solvent.
  • the attraction between the positively charged mordant, for example polyvinylbenzylamine, quaternized with methyl chloride, and the negative species such as a sulfonate substituted dye molecule, is often described as ionic in nature, but in fact, the positively charged mordant has a negative counter ion (the chloride ion) and the negatively charged dye has a positive counter ion (the sodium ion) so there is no net ionic charge available for mordanting.
  • the actual driving force for the mordanting reaction is the loss of waters of hydration when the mordanting reaction occurs. The release of the waters of hydration results in a net increase in the entropy of the system, which is the driving force for the mordanting reaction.
  • mordanting of the metal particles from the metal sol on the nucleic acid molecule is carried out by the attraction of positively charged metal in the metal sol to the negatively charged phosphate groups on the nucleic acid molecule.
  • a positively charged metal sol particle neutralizes the negative charge on a phosphate group of a nucleic acid molecule
  • the several waters of hydration that are carried by both the gold sol and by the phosphate in an organized, high entropy state are released.
  • the waters of hydration are free to migrate in all directions, and are thus in a low entropy state.
  • the change in entropy of the system provides the driving force for binding the metal in the metal sol to the phosphate group of the nucleic acid molecule.
  • the metallization by mordanting of metal particles from a metal sol, and then application of a metal deposition solution is distinguishable from the metallization process taught by Braun et al. (Rapid Commun Mass Spectrom 12(18):1246-52 (1998)), which is a process of ion replacement by mass action.
  • the immobilized DNA, with sodium counter ions is flooded with excess silver nitrate solution. In simple terms, if there is a 1000 fold excess of silver over sodium ions present, then by simple statistics, 999 of 1000 sodium ions will be replaced by silver ions. The free ions are then washed away by water, leaving immobilized DNA with silver counter ions.
  • Metals contained in the metal sol can be any one of gold, silver or a mixture thereof.
  • Other metals in the metal sol may be any of the catalytically active transition metals such as platinum, palladium, rhodium, or iridium.
  • the concentration of metal in the metal sol is from about .01 mM to about 1 mM.
  • the time required for mordanting the metal sol to the nucleic acid molecule can be from about 1 minute to about 1 hour.
  • metal from a metal deposition solution is deposited onto a mordanted nucleic acid molecule.
  • the metal particles mordanted onto a nucleic acid molecule from a metal sol become catalysts for the further deposition of metal onto the nucleic acid molecule at the mordanted sites.
  • the metal to be deposited in the metal deposition solution can be gold, silver, or a mixture thereof.
  • a suitable metal deposition solution in accordance with the present invention is comprised of potassium tetrachloroaurate, potassium thiocyanate, hydroquinone, and water.
  • the hydroquinone concentration in the gold deposition solution can range from about 1 mg/ml to about 10 mg/ml.
  • the amount of water used to dissolve the precipitate in the metal deposition solution can range from about 5 ml to about 100 ml, most preferably about 10 ml.
  • the present invention also relates to a method for detecting a target nucleic acid molecule in a sample.
  • This method involves providing a device for detecting the presence of a target nucleic acid molecule in a sample.
  • the device has two electrical conductors, including a first and second electrical conductor, which are not in contact with one another.
  • the device also has one or more sets of two oligonucleotide probes attached to the electrical conductors.
  • the probes are positioned such that they cannot come into contact with one another and such that a target nucleic acid molecule, which has two sequences, a first sequence complementary to a first probe attached to the first electrical conductor and a second sequence complementary to a second probe attached to the second electrical conductor, can bind to both probes.
  • the probes are contacted with a sample which may have the target nucleic acid molecule under selective hybridization conditions to permit target nucleic acid molecules, if any, present in the sample to hybridize to both of the probes.
  • a metal sol is provided. The metal sol and the device are contacted after the probes are contacted with the sample under conditions effective to mordant metal particles from the metal sol on any of the target nucleic acid molecules hybridized to the probes.
  • the mordanted target nucleic acid molecule is contacted with a metal deposition solution under conditions effective to deposit metal from the metal deposition solution on to any of the target nucleic acid molecules hybridized to the probes. It is then determined if an electrical current can be carried between the probes, the electrical current between the probes indicating the presence of the target nucleic acid molecule in the sample.
  • One aspect of the present invention involves the detection of nucleic acid sequences from a sample.
  • This method involves a sample collection method whereby bacteria, viruses, or other DNA containing species are collected and concentrated.
  • This method also incorporates a sample preparation method that involves the liberation of the genetic components. After liberating the nucleic acid, the sample is injected into a detection chip containing complementary nucleic acid probes for the target of interest. In this manner, the device may contain multiple sets of probe molecules that each recognizes a single but different nucleic acid sequence. This process ultimately involves the detection of hybridization products.
  • bacteria, viruses, or other DNA containing samples are collected and concentrated. A plurality of collection methods will be used depending on the type of sample to be analyzed.
  • Liquid samples will be collected by placing a constant volume of the liquid into a lysis buffer.
  • Airborne samples can be collected by passing air over a filter for a constant time. The filter will be washed with lysis buffer. Alternatively, the filter can be placed directly into the lysis buffer. Waterborne samples can be collected by passing a constant amount of water over a filter. The filter can then be washed with lysis buffer or soaked directly in the lysis buffer. Dry samples can be directly deposited into lysis buffer for removal of the organism of interest.
  • cell debris can be removed by precipitation or filtration. Ideally, the sample will be concentrated by filtration, which is more rapid and does not require special reagents.
  • FIGS 1 A-B show a perspective view of a system for detection of a target nucleic acid molecule from a sample.
  • This system includes a desk-top detection unit and a detection cartridge which is inserted into the desk-top unit.
  • desk-top detection unit 2 is provided with door 4 for filling reagents, control buttons 6, and visual display 10.
  • Slot 8 in desk-top detection unit 2 is configured to receive detection cartridge 12.
  • Detection cartridge 12 further contains first injection port 14 through which a sample solution can be introduced into a first chamber in cartridge 12 and second injection port 16 through which reagents can be introduced into the first chamber.
  • Figure 1C shows a schematic view of the system utilizing desk-top detection unit 2 and cartridge 12.
  • desk-top detection unit 2 contains containers 32A-C suitable for holding reagents and positioned to discharge the reagents into first chamber 20 of detection cartridge 12 through second injection port 16 and conduit 21.
  • Containers 32A-C can, for example, carry a neutralizer, a buffer, a conductive ion solution, and an enhancer. The contents of these containers can be replenished through door 4. This is achieved by making containers 32A-C sealed and disposable or by making them refillable.
  • Pump 28 removes reagents from containers 32A-C, through tubes 30A-
  • a separate pump can be provided for each of containers 32A-C so that their contents can be removed individually.
  • the necessary reagents may be held in containers inside the detection cartridge.
  • the pumps in the detection unit can force a material, such as air, water, or oil, into the detection cartridge to force the reagents from the respective containers and into the first chamber.
  • the reagents are then changed with each detection cartridge, which eliminates the buildup of salt precipitates in the detection unit.
  • Desk-top detection unit 12 is also provided with controller 38, which is in electrical communication with the electrical conductors of the detection cartridge 12 by means of electrical connector 36, to detect the presence of the target molecule in the sample. Controller 38 also operates pump 28 by way of electrical connector 34. Alternatively, separate controllers can be used for operating the pumps and the detection of target molecules. Digital coupling 40 permits controller 38 to communicate data to computer 42 which is external of desk-top detection unit 12.
  • Detection cartridge 12 contains first chamber 20 which, as noted supra, receives reagents from within desk-top detection unit 2 by way of second injection port 16 and conduit 21. A sample to be analyzed is discharged to first chamber 20 through first injection port 14 and conduit 18.
  • FIG. 2A depicts a single test structure on a detection chip suitable to be positioned in first chamber 20 of the system shown in Figures 1 A-C. According to Figure 2A, oligonucleotide probes 46 attached to spaced apart conductive fingers 44 are physically located at a distance sufficient that they cannot come into contact with one another.
  • a sample, containing a mixture of nucleic acid molecules (i.e. M1-M6), to be tested is contacted with the fabricated device on which conductive fingers 44 are fixed, as shown in Figure 2B. If a target nucleic acid molecule (i.e. Ml) that is capable of binding to the two oligonucleotide probes is present in the sample, the target nucleic acid molecule will bind to the two probe molecules. If bound, the nucleic acid molecule can bridge the gap between the two electrodes and provide an electrical connection. Any unhybridized nucleic acid molecules (i.e. M2-M6) not captured by the probes is washed away. [0040] Here, the electrical conductivity of nucleic acid molecules can be relied upon to transmit the electrical signal. Hans-Werner Fink and Christian
  • the target molecule is contacted with a metal sol under conditions effective to mordant metal particles from the metal sol on the target molecule.
  • the mordanted target molecule is then contacted with a metal deposition solution under conditions effective to deposit metal from the metal deposition solution on the target molecules hybridized to the probes.
  • the metallized target molecule can then conduct electricity across the gap between the pair of probes. As described supra, this flow of current can be sufficient to construct a simple switch, which will indicate whether or not a target nucleic acid molecule is present within a sample.
  • the detection of a target molecule using a desk-top detection system can be carried out as follows. After lysis and clarification of the sample, the sample is introduced into detection cartridge 12 through first injection port 14 and conduit 18 and into first chamber 20. Once the sample is introduced, detection cartridge 12 is inserted into slot 8 of desk-top detection unit 2 so that second injection port 16 is connected to conduit 21 and electrical connector 36 is coupled to electrical connector 25. The sample is processed in first chamber 20 containing the capture probes and electrical conductors for a period of time sufficient for detection of a target nucleic acid molecule in the sample.
  • Processing of the sample within first chamber 20 can involve neutralizing the sample, contacting the neutralized sample with a buffer, contacting the sample with a metal sol, then contacting the sample with a metal deposition solution. Molecules that are not captured are expelled from first chamber 20 through second conduit 22 and into second chamber 24.
  • the desk-top detection system can be programmed by a series of operation buttons 6 on the front of the device and the results can be seen on visual display 10.
  • the detection chip, on which conductive fingers 44 are fixed, is constructed on a support. Examples of useful support materials include, e.g., glass, quartz, and silicon as well as polymeric substrates, e.g. plastics.
  • FIGS 3 A-B show a portable detection system.
  • This system is provided with a portable unit 100 which can be in the form of a portable personal digital assistant (e.g., a Palm ® unit, 3Com Corporation, Santa Clara, CA).
  • Portable unit 100 is provided with visual display 102 and control buttons 104.
  • Slot 106 is provided to receive detection cartridge 108 having electrical connector 110.
  • FIG. 3C shows a schematic diagram of detection cartridge 108 which is used in the portable detection system of the present invention.
  • Detection cartridge 108 contains first injection port 112 in the housing through which a sample solution can be introduced.
  • Detection cartridge 108 contains a plurality of containers 128, 130, and 132 suitable for holding reagents and positioned to discharge the reagents into conduit 126 through conduit 134.
  • Containers 128, 130, and 132 can, for example, carry a neutralizer, a buffer, and a conductive ion solution.
  • Sample pre-treatment chamber 114 is positioned upstream of first chamber 122, with filter 118 being positioned between pretreatment chamber 114 and first chamber 122.
  • Conduits 116 and 120 provide a path between pretreatment chamber 114 and first chamber 122.
  • Detection cartridge 108 also contains conduit 124 that receives material from chamber 122.
  • Conduit 124 has a small diameter so that nucleic acid material is sheared as it passes from first chamber 122 to detection chamber 136.
  • Detection cartridge 108 also contains a waste chamber 140 coupled to detection chamber 136 by way of conduit 138 so that material discharged from the detection chamber 136 is received in waste chamber 140.
  • Detection cartridge 108 includes a series of electrical connectors 110 that are coupled to the electrically separated conductors in detection chamber 136, like those shown in first chamber 20 for the embodiment of Figures 1 A-C and 2.
  • the detection of a target molecule using a portable detection system can be carried out as follows. After lysis and clarification of the sample, the sample solution is introduced into detection cartridge 108 through first injection port 112. Within sample pretreatment chamber 114, cells are lysed to release cellular contents. After denaturation and deprotination, the sample can be partially purified by passing it through filter 118 and depositing the solution into chamber 122. Within first chamber 138, the neutralized target nucleic acid molecule, if present in the sample, is permitted to hybridize with the capture probes on the electrically separated conductors in first chamber 136 in substantially the same way as described above with reference to Figures 1 A-C and 2.
  • the sample is contacted with a metal sol from container 128, such that metal ions are mordanted on the target molecules that have hybridized to the capture probes on the detection chip. Additionally, the mordanted target molecules are contacted with a metal deposition solution from container 130 to deposit metal from the metal deposition solution on to the target molecules hybridized to the probes. Excess buffers and waste buffers will exit detection chamber 136 through waste tube 138 and collect in second chamber 140.
  • the portable detection system can be programmed by operation of a series of buttons 104 on the front of portable unit 100, and the results are visualized on screen 102.
  • a sample collection phase is initially carried out where bacteria, viruses, or other species are collected and concentrated.
  • the target nucleic acid molecule whose sequence is to be determined is usually isolated from a tissue sample. If the target nucleic acid molecule is genomic, the sample may be from any tissue (except exclusively red blood cells). For example, whole blood, peripheral blood lymphocytes or peripheral blood mononuclear cells ("PBMC"), skin, hair, or semen are convenient sources of clinical samples. These sources are also suitable if the target is RNA. Blood and other body fluids are also a convenient source for isolating viral nucleic acids. If the target nucleic acid molecule is mRNA, the sample is obtained from a tissue in which the mRNA is expressed.
  • PBMC peripheral blood mononuclear cells
  • RNA may be reverse transcribed to DNA, but need not be converted to DNA in the present invention.
  • a plurality of collection methods can be used depending on the type of sample to be analyzed.
  • Liquid samples can be collected by placing a constant volume of the liquid into a lysis buffer.
  • Airborne samples can be collected by passing air over a filter for a constant time.
  • the filter can be washed with lysis buffer.
  • the filter can be placed directly into the lysis buffer.
  • Waterborne samples can be collected by passing a constant amount of water over a filter.
  • the filter can then be washed with lysis buffer or soaked directly in the lysis buffer. Dry samples can be directly deposited into lysis buffer for removal of the organism of interest.
  • nucleic acids When whole cells, viruses, or other tissue samples are being analyzed, it is typically necessary to extract the nucleic acids from the cells or viruses, prior to continuing with the various sample preparation operations. Accordingly, following sample collection, nucleic acids may be liberated from the collected cells, viral coat, etc., into a crude extract, followed by additional treatments to prepare the sample for subsequent operations such as denaturation of contaminating (DNA binding) proteins, purification, filtration, and desalting. [0051] Liberation of nucleic acids from the sample cells or viruses, and denaturation of DNA binding proteins may generally be performed by physical or chemical methods.
  • chemical methods generally employ lysing agents to disrupt the cells and extract the nucleic acids from the cells, followed by treatment of the extract with chaotropic salts such as guanidinium isothiocyanate or urea, to denature any contaminating and potentially interfering proteins.
  • chaotropic salts such as guanidinium isothiocyanate or urea
  • the appropriate reagents may be incorporated within the extraction chamber, a separate accessible chamber, or externally introduced.
  • the sample of cells is flowed through a microtubular array while an alternating electric current is applied across the fluid flow.
  • a variety of other methods may be utilized within the device of the present invention to effect cell lysis/extraction, including, e.g., subjecting cells to ultrasonic agitation, or forcing cells through microgeometry apertures, thereby subjecting the cells to high shear stress resulting in rupture.
  • nucleic acids Following extraction, it is often desirable to separate the nucleic acids from other elements of the crude extract, e.g., denatured proteins, cell membrane particles, and the like. Removal of particulate matter is generally accomplished by filtration, flocculation, or the like. Ideally, the sample is concentrated by filtration, which is more rapid and does not require special reagents. A variety of filter types may be readily inco ⁇ orated into the device. Samples can be forced through filters that will allow only the cellular material to pass through, trapping whole organisms and broken cell debris. Further, where chemical denaturing methods are used, it may be desirable to desalt the sample prior to proceeding to the next step.
  • Desalting of the sample, and isolation of the nucleic acid may generally be carried out in a single step, e.g., by binding the nucleic acids to a solid phase and washing away the contaminating salts or performing gel filtration chromatography on the sample.
  • Suitable solid supports for nucleic acid binding include, e.g., diatomaceous earth, silica, or the like.
  • Suitable gel exclusion media is also well known in the art and is commercially available from, e.g., Pharmacia and Sigma Chemical. This isolation and/or gel filtration/desalting may be carried out in an additional chamber, or alternatively, the particular chromatographic media may be inco ⁇ orated in a channel or fluid passage leading to a subsequent reaction chamber.
  • the probes are preferably selected to bind with the target such that they have approximately the same melting temperature. This can be done by varying the lengths of the hybridization region. A-T rich regions may have longer target sequences, whereas G-C rich regions would have shorter target sequences.
  • Hybridization assays on substrate-bound oligonucleotide arrays involve a hybridization step and a detection step. In the hybridization step, the sample potentially containing the target and an isostabilizing agent, denaturing agent, or renaturation accelerant is brought into contact with the probes of the array and incubated at a temperature and for a time appropriate to allow hybridization between the target and any complementary probes.
  • hybridization optimizing agent refers to a composition that decreases hybridization between mismatched nucleic acid molecules, i.e., nucleic acid molecules whose sequences are not exactly complementary.
  • An isostabilizing agent is a composition that reduces the base-pair composition dependence of DNA thermal melting transitions. More particularly, the term refers to compounds that, in proper concentration, result in a differential melting temperature of no more than about 1°C. for double stranded DNA oligonucleotides composed of AT or GC, respectively.
  • Isostabilizing agents preferably are used at a concentration between 1 M and 10 M, more preferably between 2 M and 6 M, most preferably between 4 M and 6 M, between 4 M and 10 M, and, optimally, at about 5 M.
  • a 5 M agent in 2 x SSPE Sodium Chloride/Sodium Phosphate/EDTA solution
  • Betaines and lower tetraalkyl ammonium salts are examples of suitable isostabilizing agents.
  • Betaine N,N,N,-trimethylglycine; (Rees et al., Biochem. 32: 137- 144
  • betaine is zwitterionic at neutral pH and does not alter the polyelectrolyte behavior of nucleic acids while it does alter the composition-dependent stability of nucleic acids. Inclusion of betaine at about 5 M can lower the average hybridization signal, but increases the discrimination between matched and mismatched probes.
  • a denaturing agent is a composition that lowers the melting temperature of double stranded nucleic acid molecules by interfering with hydrogen bonding between bases in a double-stranded nucleic acid or the hydration of nucleic acid molecules.
  • Denaturing agents can be included in hybridization buffers at concentrations of about 1 M to about 6 M and, preferably, about 3 M to about 5.5 M.
  • Denaturing agents include formamide, formaldehyde, dimethylsulfoxide (“DMSO”), tetraethyl acetate, urea, guanidine thiocyanate (“GuSCN”), glycerol and chaotropic salts.
  • Chaotropic salt refers to salts that function to disrupt van der Waal's attractions between atoms in nucleic acid molecules. Chaotropic salts include, for example, sodium trifluoroacetate, sodium tricholoroacetate, sodium perchlorate, and potassium thiocyanate.
  • a renaturation accelerant is a compound that increases the speed of renaturation of nucleic acids by at least 100-fold. They generally have relatively unstructured polymeric domains that weakly associate with nucleic acid molecules. Accelerants include heterogenous nuclear ribonucleoprotein ("hnRP") Al and cationic detergents such as, preferably, cetyltrimethylammonium bromide (“CTAB”) and dodecyl trimethylammonium bromide (“DTAB”), and, also, polylysine, spermine, spermidine, single stranded binding protein (“SSB”), phage T4 gene 32 protein, and a mixture of ammonium acetate and ethanol. Renaturation accelerants can be included in hybridization mixtures at concentrations of about 1 ⁇ M to about 10 mM and, preferably, 1 ⁇ M to about 1 mM. The CTAB buffers work well at concentrations as low as 0.1 mM.
  • hybridization buffers Addition of small amounts of ionic detergents (such as N-lauroyl- sarkosine) to the hybridization buffers can also be useful. LiCl is preferred to NaCl. Hybridization can be at 20°-65°C, usually 37°C to 45°C for probes of about 14 nucleotides. Additional examples of hybridization conditions are provided in several sources, including: Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, N.Y. (1989); and Berger and Kimmel, "Guide to Molecular Cloning Techniques," Methods in Enzymology, Volume 152, Academic Press, Inc., San Diego, Calif. ( 1987); Young and Davis, Proc. Natl Acad. Sci. USA, 80: 1194 (1983), which are hereby inco ⁇ orated by reference in their entirety. [0063] In addition to aqueous buffers, non-aqueous buffers may also be used.
  • ionic detergents such as N
  • sample and hybridization reagents are placed in contact with the array and incubated. Contact can take place in any suitable container, for example, a dish or a cell specially designed to hold the probe array and to allow introduction and removal of fluids. Generally, incubation will be at temperatures normally used for hybridization of nucleic acids, for example, between about 20°C and about 75°C, e.g., about 25°C, about 30°C, about 35°C, about 40°C, about 45°C, about 50°C, about 55°C, about 60°C, or about 65°C. For probes longer than about 14 nucleotides, 37-45°C is preferred.
  • hybridization is carried out at a temperature at or between ten degrees below the melting temperature and the melting temperature. More preferred, hybridization is carried out at a temperature at or between five degrees below the melting temperature and the melting temperature.
  • the target is incubated with the capture probes for a time sufficient to allow the desired level of hybridization between the target and any complementary capture probes.
  • the electrically separated conductors are washed with the hybridization buffer, which also can include the hybridization optimizing agent. These agents can be included in the same range of amounts as for the hybridization step, or they can be eliminated altogether.
  • functional for oligonucleotides, typically an -OH
  • a nucleoside building block itself protected with a photo-removable protecting group (at the 5'-OH)
  • the process can be repeated, using different masks or mask orientations and building blocks, to place probes on a substrate.
  • the probes are attached to the leads through spatially directed oligonucleotide synthesis.
  • Spatially directed oligonucleotide synthesis may be carried out by any method of directing the synthesis of an oligonucleotide to a specific location on a substrate.
  • Methods for spatially directed oligonucleotide synthesis include, without limitation, light-directed oligonucleotide synthesis, microlifhography, application by ink jet, microchannel deposition to specific locations and sequestration with physical barriers. In general, these methods involve generating active sites, usually by removing protective groups, and coupling to the active site a nucleotide which, itself, optionally has a protected active site if further nucleotide coupling is desired.
  • the lead-bound oligonucleotides are synthesized at specific locations by light-directed oligonucleotide synthesis which is disclosed in U.S. Patent No. 5,143,854, Published PCT Application Serial No. WO 92/10092, and Published PCT Application Serial No. WO 90/15070, which are hereby inco ⁇ orated by reference in their entirety.
  • the surface of a solid support modified with linkers and photolabile protecting groups is illuminated through a photolithographic mask, yielding reactive hydroxyl groups in the illuminated regions.
  • a 3'-O-phosphoramidite-activated deoxynucleoside (protected at the 5'-hydroxyl with a photolabile group) is then presented to the surface and coupling occurs at sites that were exposed to light. Following the optional capping of unreacted active sites and oxidation, the substrate is rinsed and the surface is illuminated through a second mask, to expose additional hydroxyl groups for coupling to the linker. A second 5'-protected, 3'-O-phosphoramidite-activated deoxynucleoside (C-X) is presented to the surface. The selective photodeprotection and coupling cycles are repeated until the desired set of probes are obtained. Photolabile groups are then optionally removed, and the sequence is, thereafter, optionally capped. Side chain protective groups, if present, are also removed. Since photolithography is used, the process can be miniaturized to specifically target leads in high densities on the support.
  • the protective groups can, themselves, be photolabile. Alternatively, the protective groups can be labile under certain chemical conditions, e.g., acid.
  • the surface of the solid support can contain a composition that generates acids upon exposure to light. Thus, exposure of a region of the substrate to light generates acids in that region that remove the protective groups in the exposed region.
  • the synthesis method can use 3'-protected 5'-O-phosphoramidite-activated deoxynucleoside. In this case, the oligonucleotide is synthesized in the 5' to 3' direction, which results in a free 5' end.
  • the probes may be targeted to the electrically separated conductors by using a chemical reaction for attaching the probe or nucleotide to the conductor which preferably binds the probe or nucleotide to the conductor rather than the support material.
  • the probe or nucleotide may be targeted to the conductor by building up a charge on the conductor which electrostatically attracts the probe or nucleotide.
  • Nucleases can be used to remove probes which are attached to the wrong conductor. More particularly, a target nucleic acid molecule may be added to the probes. Targets which bind at both ends to probes, one end to each conductor, will have no free ends and will be resistant to exonuclease digestion.
  • probes which are positioned so that the target cannot contact both conductors will be bound at only one end, leaving the molecule subject to digestion.
  • improperly located probes can be removed while protecting the properly located probes.
  • the target nucleic acid molecule can be removed and the device is ready for use.
  • the capture probes can be formed from natural nucleotides, chemically modified nucleotides, or nucleotide analogs, as long as they have activated hydroxyl groups compatible with the linking chemistry.
  • RNA or DNA analogs comprise but are not limited to 2'-O-alkyl sugar modifications, methylphosphonate, phosphorothioate, phosphorodithioate, formacetal, 3'-fhioformacetal, sulfone, sulfamate, and nitroxide backbone modifications, amides, and analogs, where the base moieties have been modified.
  • analogs of oligomers may be polymers in which the sugar moiety has been modified or replaced by another suitable moiety, resulting in polymers which include, but are not limited to, polyvinyl backbones (Pitha et al., "Preparation and Properties of Poly (I-vinylcytosine),” Biochim. Biophys. Acta 204:381-8 (1970); Pitha et al., “Poly(l-vinyluracil): The Preparation and Interactions with Adenosine Derivatives," Biochim. Biophys.
  • the capture probes can contain the following exemplary modifications: pendant moieties, such as, proteins (including, for example, nucleases, toxins, antibodies, signal peptides and poly-L-lysine); intercalators (e.g., acridine and psoralen), chelators (e.g., metals, radioactive metals, boron and oxidative metals), alkylators, and other modified linkages (e.g., alpha anomeric nucleic acids).
  • proteins including, for example, nucleases, toxins, antibodies, signal peptides and poly-L-lysine
  • intercalators e.g., acridine and psoralen
  • chelators e.g., metals, radioactive metals, boron and oxidative metals
  • alkylators e.g., alpha anomeric nucleic acids
  • Such analogs include various combinations of the above-mentioned modifications involving linkage groups and/or structural modifications of the sugar or base for the pu ⁇ ose of improving RNAseH -mediated destruction of the targeted RNA, binding affinity, nuclease resistance, and or target specificity.
  • the present invention can be used for numerous applications, such as detection of pathogens. For example, samples may be isolated from drinking water or food and rapidly screened for infectious organisms. The present invention may also be used for food and water testing. In recent times, there have been several large recalls of tainted meat products.
  • the detection system of the present invention can be used for the in-process detection of pathogens in foods and the subsequent disposal of the contaminated materials.
  • the present invention can be used for real time detection of biological warfare agents.
  • the device With the recent concerns of the use of biological weapons in a theater of war and in terrorist attacks, the device could be configured into a personal sensor for the combat soldier or into a remote sensor for advanced warnings of a biological threat.
  • the devices which can be used to specifically identify the agent can be coupled with a modem to send the information to another location.
  • Mobile devices may also include a global positioning system to provide both location and pathogen information.
  • the present invention may be used to identify an individual.
  • a series of probes, of sufficient number to distinguish individuals with a high degree of reliability, are placed within the device.
  • Various polymo ⁇ hism sites are used.
  • the device can determine the identity to a specificity of greater than one in one million, more preferred is a specificity of greater than one in one billion, even more preferred is a specificity of greater than one in ten billion.
  • Silicon wafers were oxidized to provide a 500 nm silicon oxide electrically insulating top layer.
  • the wafer was then coated with positive photoresist, imaged with a mask having openings where metal is desired, and developed to give open areas where metal is desired.
  • the wafer was then placed in the vacuum evaporator, and about 50 nm of chromium followed by about 50 nm of gold was evaporated onto the wafer.
  • the wafer was then developed in photoresist developer to lift off the unwanted metal areas, leaving a pattern of interdigitated 1 micron wires with 1 micron spaces between them, along with lead wires connecting the interdigitated wires to electrical interrogation pads.
  • the wafer was then diced on a diamond saw into individual chips, each chip having 14 features, each feature consisting of 100 left connected wires 1 micron thick and 200 microns long interdigitated with 100 right connected wires, also 1 micron thick and 200 microns long, with 1 micron spaces between the wires. Electrical measurements were taken at this point to be sure there was no electrical conductivity between the right and left hand wires. [0081] The chips were then cleaned in a solution that is 1 part concentrated ammonium hydroxide to 10 parts 30% hydrogen peroxide. The chips were then washed with several changes of E-pure water. The chips were then dried under a stream of dry nitrogen.
  • the blocking solution (1 mM 6-mercapto, 1-hexanol in E pure water) was poured over the chips and incubated for 3 hours at room temperature. The blocking solution was poured off and the chips rinsed with several changes of E pure water and dried under a stream of dry nitrogen gas.
  • Example 4 Hybridization of Target DNA from a Sample
  • the sample was incubated for 10 minutes at 95°C to denature the DNA.
  • 4-5 chips were placed in a row in a sterile 500 ml centrifuge bottle. They were placed face down with the broad side of the chips parallel to the mouth of the bottle. 3 ml of the denatured template DNA was gently poured into the centrifuge bottle containing the microchips.
  • the bottle was immediately placed on a rocker platform in a 65°C incubator.
  • the bottle was positioned so that the liquid inside the bottle flowed from top to bottom of the bottle when the platform was rocked.
  • the bottle was incubated for 1 hour at 65°C with gentle rocking.
  • the chips were washed once with citrate buffer at 65°C, and rinsed once with pure water.
  • Nanogold® (#2022) (Nanoprobes, Yaphank, NY) in 1.5 ml of pure water. This solution was spotted on the features of the hybridized chip and incubated at room temperature for 15 minutes. The chip was then rinsed with pure water and dried under a stream of nitrogen.
  • Example 6 Preparation of a Metal Deposition Solution
  • a solution of 230 mg of potassium tetrachloroaurate in 10 ml of water was prepared.
  • a solution of 600 mg of potassium thiocyanate in 10 ml of water was prepared.
  • 1 ml of each of the two solutions was mixed.
  • the mixture turned dark red and a brick red precipitate formed.
  • the mixture was centrifuged for 5 minutes at 1300 ⁇ m to separate the precipitate.
  • the supernatant liquid was decanted, and the precipitate was dissolved by mixing with 10 ml of water to give a colorless solution that was then filtered through a 0.2 micron filter. This was called the metal deposition solution.
  • a solution of 230 mg of potassium tetrachloroaurate in 10 ml of water was prepared.
  • a solution of 600 mg of potassium thiocyanate in 10 ml of water was prepared.
  • One ml of each of the two solutions was mixed.
  • the mixture turned dark red and a brick red precipitate formed.
  • the mixture was centrifuged for 5 minutes at 1300 ⁇ m to separate the precipitate.
  • the supernatant liquid was decanted, and the precipitate was dissolved by mixing with 10 ml of water to give a colorless solution that was then filtered through a 0.2 micron filter. This was called the metal deposition solution.
  • a solution of 25 mg of hydroquinone in 5 ml water was prepared. The solution was filtered through a 0.2 micron filter.
  • Example 10 - Control All of the steps of Examples 1-7 were repeated, except the hybridization step (Example 4) was omitted. Electrical measurements showed that half (50%) of the features were shorted and the other half had a resistance of greater than 10 megaohms.

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Abstract

L'invention concerne un procédé de métallisation de molécule d'acides nucléiques par l'établissement d'un sol métallique, le mordançage des particules métalliques de ce sol sur la molécule d'acides nucléiques, puis le contact entre ladite molécule mordancée et une solution de dépôt métallique, pour le dépôt de métal sur cette molécule. On décrit également un procédé de détection de molécule d'acides nucléiques cible dans un échantillon au moyen de molécules d'acides nucléiques métallisées.
PCT/US2004/043530 2003-12-30 2004-12-22 Metallisation d'acides nucleiques par sols metalliques, et utilisations correspondantes Ceased WO2005065290A2 (fr)

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