CN1878874A - Direct SNP detection with unamplified DNA - Google Patents

Abstract

This invention provides a method for detecting target nucleic acid molecules in a sample, wherein the sample contains nucleic acid molecules with higher biological complexity than the amplified nucleic acid molecules. Specifically, this invention provides a method and probe for detecting single nucleotide polymorphisms (SNPs) in a sample, wherein the sample contains nucleic acid molecules with higher biological complexity than the amplified nucleic acid molecules.

Description

Direct SNP detection using non-amplified DNA

This application is related to, and claims priority from, U.S. Provisional Application Nos. 60/432,772 and 60/433,442, filed December 12, 2002, the disclosures of which are incorporated herein by reference.

technical field

The present invention relates to methods for detecting target nucleic acid molecules in a sample comprising nucleic acid molecules of higher biological complexity than amplified nucleic acid molecules, for example in genomic DNA. In particular, the present invention relates to methods and probes for detecting SNPs using nanoparticle-labeled probes. The present invention also relates to methods of detecting biological organisms, in particular bacterial pathogens such as staphylococcal DNA, and detecting antibiotic resistance genes, such as the mecA gene conferring resistance to the antibiotic methicillin, in a sample.

Background technique

Single nucleotide polymorphisms (SNPs), or single base variations, between genomic DNA observed in different individuals not only form the basis of genetic diversity, but are also considered markers of disease predisposition and can be used to better understand Improve disease control, increase understanding of disease conditions, and ultimately facilitate the discovery of more effective medicines. Therefore, a great deal of effort is being made towards the common goal of developing methods that can identify SNPs rapidly and reliably. Due to the inherent complexity of human genomic DNA (haploid genome = 3 x ¹⁰⁹ bp) and the associated sensitivity requirements, most of these efforts have required targeted amplification by methods such as PCR. The ability to detect SNPs directly in human genomic DNA will simplify detection and eliminate errors associated with target amplification in SNP identification.

Single nucleotide polymorphisms can be identified using a number of methods, including DNA sequencing, restriction enzyme analysis, or specific site hybridization. However, high-throughput genome-wide screening of SNPs and mutations requires the ability to analyze multiple loci simultaneously with high precision and sensitivity. To improve sensitivity and specificity, current high-throughput methods for the detection of single nucleotides rely on steps involving amplification of target nucleic acid samples, usually by polymerase chain reaction (PCR) (see, e.g., Nikiforov et al., published in 1997 U.S. Pat. No. 5,679,524, October 21, 1998; McIntosh et al., PCT Application WO 98/59066, published December 30, 1998; Goelet et al., PCT Application WO 95/12607, published May 11, 1995 ; Wang et al., 1998, Science 280: 1077-1082; Tyagi et al., 1998, Nature Biotechnol.16: 49-53; Chen et al., 1998, Genome Res.8: 549-556; Pastinen et al., 1996, Clin.Chem. 42:1391-1397; Chen et al., 1997, Proc.Natl.Acad.Sci.94:10756-10761; Shuber et al., 1997, Hum.Mol.Gen.6:337-347; 7:389-398; Livak et al., Nature Genet. 9:341-342; Day and Humphries, 1994, Anal. Biochem. 222:389-395). There are two main reasons why PCR amplification is necessary for traditional SNP-based hybridization: First, when obtaining total human DNA with 3,000,000,000 base pairs per haploid genome, targets containing SNP loci Sequences represent only a very small fraction of total DNA. For example, a 20 base pair target sequence represents only 0.00000033% of the total DNA (a normal genome has two copies of the target sequence, but they have different SNP sites and are therefore considered different sites). Therefore, common DNA samples of a few microorganisms are insufficient for many existing techniques due to insufficient sensitivity. However, the more important reason is that the hybridization of oligonucleotides short enough to allow discrimination of single bases to a 20-base target sequence hybridizes not exclusively to the target region, but to a lesser extent. other regions of the genome. Due to the overwhelming amount of non-target DNA, non-specific hybridization creates so much background that it masks specific signals. Therefore, amplification of a target region is a necessary step to significantly reduce non-specific sequences. This amplification step is called "complexity reduction". However, the fidelity of PCR technology is limited. Combinations of PCR primer pairs tend to produce spurious reaction products or are unsuccessful in some specific regions. Additionally, when non-target sequences are replicated, or errors are introduced into the target sequence due to misbinding, the number of errors in the final product increases exponentially with each cycle of PCR amplification. Thus, PCR errors can be a fundamental shortcoming when searching for rare variants in nucleic acid populations.

Finally, the disadvantage of using target amplification is that each SNP locus must be amplified separately. And since there are potentially millions of SNPs in the human genome, this becomes an impossible task. Even though methods and strategies for amplification revolve around the issue of SNP-site-specific amplification, the current state of the art can only simultaneously identify a small percentage (less than 0.1%) of the total SNPs (see, e.g., Kennedy et al., 2003, Nature Biotechnol. 21:1233-1237). Eric Lander of the Whitehead Institute for Biomedical Research and one of the leaders of the Human Genome Project pointed out that discarding target amplification is one of the most significant challenges in genome-wide SNP screening (see Lander E.1999, Nature Genetics Suppl. 21:3-4). Therefore, there is still a need in the prior art for more sensitive, effective, and low-cost methods for detecting SNPs in samples, such methods do not require target amplification or simplify the complexity.

The identification of DNA mutations is also important for the identification of microorganisms (see Edwards et al., J. Clin. Micro. 39:3047-3051). For example, the Staphylococcus genus includes at least 38 different species, most of which have been identified in nosocomial infections (Edwards et al., J. Clin. Micro. 39:3047-3051). Therefore, the rapid identification and speciation of microorganisms is important to identify the source of infection, which helps to determine the treatment of patients, as well as to identify the outbreak of infection and the cross transmission of nosocomial pathogens in an epidemiological sense ( Olive and Bean, 1999, J. Clin. Micro. 37:1661-1669). Traditional methods of identifying bacteria based on biochemical tests are often time-consuming (1>day) and often fail to accurately identify specific species (Hamels et al., 2001, Biotechniques 31: 1364-1372). Therefore, a lot of work is focused on the development of faster, more accurate, and less expensive identification methods based on nucleic acid sequence identification of specific bacterial species, especially nosocomial pathogens such as Staphylococcus. Microorganisms of the same family or genus contain phylogenetically conserved genes encoding the same protein (Hamels et al., 2001, Biotechniques 31: 1364-1372). Although gene sequences from the same family are often highly conserved, species-specific sequence mutations have been identified in various genes such as 16S rRNA. Oligonucleotide probes targeting the variable region of the 16S rRNA gene have been developed for real-time PCR detection to identify a variety of coagulase-negative and positive staphylococcal species (Edwards et al., J.Clin.Micro.39:3047-3051 ).

Additionally, microarrays have been developed to identify Staphylococcus genus, species and antibiotic resistance by PCR amplified femA gene sequences (Hamels et al., 2001, Biotechniques 31:1364-1372). The microarray contains oligonucleotide probes that identify species-specific sequence variations (sequence variations of three or more bases) in the femA gene associated with five clinically relevant Staphylococcus species (S. aureus (S.aureus, S.epidermidis, S.haemolyticus, S.hominis, S.saprophyticus), and target the same gene Oligonucleotide probes of conserved regions were used for identification of Staphylococcus species. However, a major disadvantage of microarray and real-time PCR-based assays is the need for PCR, which makes them less than ideal from both a clinical and cost standpoint (see discussion of PCR for SNP identification above). Therefore, there is still a need in the prior art for more sensitive, effective, and lower-cost methods for detecting and analyzing biological microorganisms in samples, such methods do not require target amplification or complexity reduction.

Summary of the invention

The present invention provides methods for detecting a target nucleic acid sequence in a sample, wherein the sample contains nucleic acid molecules of higher biological complexity than amplified nucleic acid molecules, the target nucleic acid sequence differs from known nucleic acid sequences by at least one single nucleotide. For example, a single nucleotide difference would be a single nucleotide polymorphism.

In one aspect, a method for detecting a target nucleic acid sequence in a sample that does not include upfront target amplification or complexity reduction comprises the steps of: a) providing an addressable substrate bound to a capture oligonucleotide, wherein the capture oligonucleotide has A sequence complementary to at least a part of the first part of the target nucleic acid sequence; b) providing a detection probe comprising a detection oligonucleotide, wherein the detection oligonucleotide has a second part of the target nucleic acid sequence in step (a) at least a partially complementary sequence; c) subjecting the sample to the substrate and the probe under conditions under which the capture oligonucleotide is capable of effectively hybridizing to a first portion of the target nucleic acid sequence, and the detection probe is capable of effectively hybridizing to a second portion of the target nucleic acid sequence contacting the probe; and d) detecting whether the capture oligonucleotide and detection probe hybridize to the first and second portions of the target nucleic acid sequence.

In another aspect, a method of detecting a target nucleic acid sequence in a sample that does not involve upfront target amplification or complexity reduction comprises the steps of: a) providing an addressable substrate bound to a plurality of capture oligonucleotides, wherein the capture oligonucleotides The acid has a sequence complementary to one or more parts of the target nucleic acid sequence; b) providing a detection probe comprising a detection oligonucleotide, wherein the detection oligonucleotide has a A sequence that is complementary to one or more portions of the target nucleic acid sequence recognized by the nucleotide; c) when the capture oligonucleotide is capable of efficiently hybridizing to one or more portions of the target nucleic acid sequence, and the detection probe is capable of interacting with the non-captured oligonucleotide contacting the sample with the substrate and the detection probe under conditions effective to hybridize to one or more portions of the target nucleic acid sequence recognized by the nucleotide; and d) detecting whether the capture oligonucleotide and the detection probe hybridize to the target nucleic acid sequence.

The invention also provides methods for identifying single nucleotide polymorphisms in a sample comprising nucleic acid molecules of greater biological complexity than amplified nucleic acid molecules.

In one aspect, a method of identifying a single nucleotide polymorphism in a sample that does not involve up-front target amplification or complexity reduction comprises the steps of: a) providing an addressable substrate bound to at least one capture oligonucleotide, wherein said At least one capture oligonucleotide has a sequence complementary to at least a portion of the nucleic acid target comprising a specific polymorphism; b) providing a detection probe combined with a detection oligonucleotide, wherein the detection oligonucleotide has a (a) a sequence complementary to at least a portion of the nucleic acid target; c) allowing the sample to interact with the substrate and contacting the detection probe; and d) detecting whether the capture oligonucleotide and the detection probe hybridize to the nucleic acid target.

In another aspect, a method of identifying single nucleotide polymorphisms in a sample that does not involve up-front target amplification or complexity reduction comprises the steps of: a) providing an addressable substrate bound to a plurality of capture oligonucleotides, wherein the capture The oligonucleotide has a sequence complementary to multiple portions of the nucleic acid target, each portion comprising a specific polymorphism; b) providing a detection probe comprising a detection oligonucleotide, wherein the detection oligonucleotide has a A sequence complementary to at least a portion of the nucleic acid target of step (a), the nucleic acid target is not recognized by the capture oligonucleotide on the substrate; c) when the capture oligonucleotide is capable of effectively hybridizing to multiple portions of the nucleic acid target , and contacting the sample with the substrate and the detection probe under conditions under which the detection probe effectively hybridizes to the nucleic acid target; and d) detecting whether the capture oligonucleotide and the detection probe hybridize to the nucleic acid target.

In one embodiment, the nucleotide difference or single nucleotide polymorphism of the target nucleic acid can be recognized by the capture oligonucleotide or detection oligonucleotide bound to the substrate.

In another embodiment, the target nucleic acid molecule in the sample comprises genomic DNA, genomic RNA, expressed RNA, plasmid DNA, mitochondrial or other cellular organelle DNA, free cellular DNA, viral DNA or viral RNA, or both or A mixture of two or more.

In one embodiment, the substrate used in the methods of the invention may comprise a number of capture oligonucleotides, each capture oligonucleotide capable of recognizing one or more different single nucleotide polymorphisms or nucleotides Differentially, a sample may contain more than one nucleic acid target, each nucleic acid target containing a different SNP or nucleoside difference capable of hybridizing to one of many capture oligonucleotides. In addition, the methods of the invention may provide one or more types of detection probes, each type of detection probe having incorporated detection oligonucleotides capable of hybridizing to a different nucleic acid target.

In one embodiment, a sample can be contacted with a detection probe such that nucleic acid targets present in the sample hybridize to detection oligonucleotides on the detection probe, and the nucleic acid target bound to the detection probe can then be The object is contacted with the substrate to hybridize the nucleic acid target to the capture oligonucleotide on the substrate. Optionally, the sample can be contacted with a substrate to allow nucleic acid targets in the sample to hybridize to capture oligonucleotides, and then the nucleic acid targets bound to the capture oligonucleotides can be contacted with detection probes to allow the nucleic acid targets to hybridizes to the detection oligonucleotides on the detection probes. In another embodiment, the sample can be contacted with the probe probe and the substrate simultaneously.

In another embodiment, the detection oligonucleotide can comprise a detectable label. The label can be, for example, fluorescent, luminescent, phosphorescent, radioactive, or a nanoparticle, and the probe oligonucleotide can be attached to a dendrimer, molecular aggregate, Quantum dots, or beads. The label can be detected by, for example, photonic, electronic, acoustic, photoacoustic, gravitational, electrochemical, electro-optical, mass spectrometric, enzymatic, chemical, biochemical, or physical means.

In one embodiment, the detection probe may be a nanoparticle probe bound to a detection oligonucleotide. Nanoparticles can be made of precious metals such as gold or silver. Nanoparticles can be detected using, for example, optical or flatbed scanners. The scanner can be connected to a computer, and software capable of calculating the gray value is installed on the computer, and the calculated gray value provides a quantitative value of the detected nucleic acid amount. In place of nanoparticles made of gold, silver or other metals that enable autometallography, substrates bound to nanoparticles in the form of target nucleic acid molecules can be detected with high sensitivity by silver staining. Optionally, the substrate bound to the nanoparticles can be detected by detecting light scattered by the nanoparticles.

In another embodiment, oligonucleotides attached to a substrate can be placed between two electrodes, the nanoparticles can be made of an electrically conductive material, step (d) of the method of the invention can include detecting the conductivity Variety. In another embodiment, a plurality of oligonucleotides each capable of recognizing a different target nucleic acid sequence are attached to a substrate in an array of spots, each oligonucleotide spot being located between two electrodes, the nanoparticles May be made of an electrically conductive material, and step (d) of the method of the invention may comprise detecting a change in electrical conductivity. The electrodes can be made of gold, for example, and the nanoparticles are also made of gold. Optionally, the substrate can be contacted with a silver stain to create a change in conductivity.

In another embodiment, the methods of the invention can be used to distinguish between two or more species of the same genus. In one aspect, the species can differ by two or more discrete nucleotides. On the other hand, the species may differ by two or more consecutive nucleotides.

In one embodiment, the target nucleic acid sequence of the present invention may be a part of a staphylococcal gene. In one aspect of this embodiment scheme, the Staphylococcus can be, for example, Staphylococcus aureus (S.aureus), Staphylococcus hemolyticus (S.haemolyticus), Staphylococcus epidermidis (S.epidermidis), Staphylococcus Lyonnais (S.lugdunensis), human Type Staphylococcus (S.hominis), Saprophytic Staphylococcus (S.saprophyticus). Therefore, the method of the present invention can be used in the speciation analysis of Staphylococci (eg: distinguishing between different species of Staphylococci).

In another embodiment, the target nucleic acid sequence of the present invention may be a part of the mec A gene. Therefore, the method of the present invention can be used to identify methicillin-resistant strains.

In another embodiment of the present invention, target nucleic acid sequence, capture oligonucleotide, and/or detection oligonucleotide can comprise SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO : 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO : 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO : 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO : 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, or a sequence listed in SEQ ID NO: 78 .

Specific preferred embodiments of the invention will be apparent from the following more detailed description of some preferred embodiments and the claims.

Description of drawings:

Figure 1 is a schematic diagram of the one-step hybridization method of the present invention.

Fig. 2 is a schematic diagram of the two-step hybridization method of the present invention.

Figure 3 schematically shows a hybrid complex of a nanoparticle-labeled detection probe, a wild-type or mutant capture probe bound to a substrate, and a wild-type target. To detect SNPs, the test is performed under appropriate experimental conditions that preserve complexes with perfect matches (left) and prevent the formation of complexes containing mismatches (right).

Fig. 4 shows the factor V gene ( 1691 G->A) SNP detection. Part (c) is a summary graph of the detection signal intensity analysis of human genomic DNA and non-specific salmon sperm DNA in the presence of wild-type or mutant capture probes.

Figure 5 shows the importance of adjusting hybridization conditions to enable the method of the present invention to discriminate between two target nucleic acids that differ by one nucleotide (SNP site).

Figure 6(a), (b) and (c) show that arrays (capture probe sequences) and hybridization conditions can be designed such that more than one SNP type can be detected on the same array and under the same hybridization conditions, independent of Under conditions of input DNA, SNP discrimination between wild-type and mutant DNA is possible.

Figure 7 shows that on the CodeLink(R) slide, under different formamide concentration conditions, the SNP detection of the factor V mutant gene (1691 G->A) is carried out with non-amplified human genomic DNA (part (a)) by hybridization, Wild-type and mutant Factor V gene capture probes are arrayed on CodeLink(R) slides. Part (b) is a graph summarizing the detection signal intensity analysis of human genomic DNA after hybridization at different formamide concentrations in the presence of wild-type or mutant capture probes.

Figure 8 shows that under optimally adjusted conditions, human wild-type DNA only produces a signal on the wild-type probe, while human mutant DNA only produces a signal on the mutant capture probe.

Figures 9(a)-(d) show quantitative data for ideal (intermediate) hybridization conditions in Figure 8 .

Figures 10(a) and (b) show that SNPs can be discriminated using very small amounts (less than 1 mg) of total human DNA. Figure 10 also shows the importance of capture oligonucleotide design and proper matching under stringent conditions for the length and nucleotide composition of the capture (and detection) probes.

Figures 11 (a) and (b) show the results of SNP detection in genomic DNA in 10 individual hybridizations on one slide using the method of the present invention. There was no overlap in standard deviations of net signal intensities for matches and mismatches across 10 hybridizations, meaning that for each hybridization reaction, the SNP genotype of the input DNA could be reliably determined.

Figure 12(a) and (b) show the results of multiple SNP identification in the whole genome DNA using the method of the present invention, in which the genotypes of Factor V, Factor II and MTHFR genes were detected.

Figure 13(a) and (b) show the results of multiplex SNP identification in the whole genomic DNA of patient sample GM16028, which shows that the method of the present invention identifies heterozygous SNP genes for Factor V, Factor II and MTHFR genes in a single individual type ability.

Figure 14(a) and (c) show the results of multiplex SNP detection in the whole genomic DNA of patient sample GM00037, which shows that the method of the present invention identifies a single individual as wild for one gene (factor V in this case), The ability to be heterozygous for another gene (factor II in this case) and mutated for a third gene (MTHFR in this case).

Figures 15(a)-(f) show the results of three different investigators performing the method of the invention on two separate patient samples.

Figure 16(a)-(b) shows the results of specific detection of the mecA gene derived from Staphylococcus genomic DNA isolated on Methicillin-resistant (mecA+) Staphylococcus aureus cells with mecA4-labeled gold nanoparticles as detection probes. Staphylococcal genomic DNA isolated from methicillin-sensitive (mecA-) S. aureus cells was used as a negative control. A known amount of PCR amplified mecA gene (281 base-pair fragment labeled MRSA 281bp) was used as a positive control. Part (a) shows a series of scanned images from microarray wells containing variable amounts of methicillin-resistant genomic DNA targets (75-300 million copies) with the same positive and negative control samples. Part (b) is a graph showing analysis of sample data. The net signal of methicillin-resistant S. aureus genomic DNA was plotted by subtracting the signal of the corresponding negative control spot. In all bars, the horizontal black line represents three standard deviations relative to negative control spots containing methicillin-susceptible S. aureus genomic DNA. This figure shows the specific detection of mec A from total bacterial genomic DNA.

Figure 17 illustrates staphylococcal speciation assays using PCR amplicons (amplicons) or genomic DNA derived from S. aureus and S. epidermidis (ATCC numbers 700699 and 35984, respectively). To detect total genomic DNA, DNA samples were fragmented by sonication prior to array hybridization. Part (a) is a series of scanned images from microarray wells containing Tuf 372bp amplicon or genomic DNA (300ng, ~8.0E7 copies). Water (no target) was used as a control. The array plate includes Tuf 3 and Tuf 4 capture probes bound thereto. Gold nanoparticle-labeled Tuf 2 probes were used as detection probes. Part (b) provides a graph representing the analysis of the sample data shown in part (a). Horizontal black lines represent three standard deviations from background. (c) Partial Tuf 372bp amplicon or genomic DNA (8.0E7 copies). The array plate includes Tuf 5 and Tuf 6 capture probes bound thereto. Part (d) provides graphs representing the analysis of the sample data shown in part (c). Horizontal black lines represent three standard deviations from background.

Figure 18(a)-(c) provides the 281 base pair S. aureus mecA, the 450 base pair S. aureus coa, the 142 base pair gold used in Examples 4-6. Sequences of the PCR amplicons of S. aureus Tuf, 372 base pairs of S. aureus Tuf, and 372 base pairs of S. epidermidis Tuf.

Figure 19(a)-(j) exemplifies staphylococcal speciation analysis and mecA gene detection using PCR amplified targets from commercially available staphylococcus strains ATCC 35556, ATCC 35984, ATCC 12228, ATCC 700699, and ATCC 15305. Parts (a), (c), (e), (g) and (i) are a series of scanned images from microarray wells containing any of the 16S, Tuf or mecA genes representing five genomic samples PCR product. Parts (b), (d), (f) and (h) are a series of graphs representing the analysis of data from 5 samples. In all bars, the horizontal black line represents three standard deviations from the background.

Figure 20(a)-(f) exemplifies staphylococcal speciation analysis and mec A detection using sonicated genomic DNA targets from commercially available staphylococcal strains ATCC 35984, ATCC 700699 and ATCC 12228. Parts (a), (c) and (e) are a series of scanned images of microarray wells containing genomic DNA from any of ATCC 35984, ATCC 700699 or ATCC12228. Array plates include 16S 12, mecA 6, Tuf 3, Tuf 4, Tuf 10 capture probes combined with negative hybridization controls. AuNP-labeled 16S13, mecA4 and Tuf2 probes were used as detection probes. Parts (b), (d) and (f) are a series of graphs representing the analysis of data from three samples. In all bars, the horizontal black line represents three standard deviations from the background.

Figure 21 is a graph showing the sensitivity margins for mec A gene detection with genomic DNA targets. Using the sequence in Table 3, under the conditions of 5x SCC, 0.05% Tween 20, 0.01% BSA, 15% v/v formamide and 200pM nanoparticle probe, 45°C for 1.5 hours, to perform the mec A gene of ATCC700699 genomic sample Detection data analysis. Figure 21 shows a detection limit of 330 fM in a 50 μl reaction (34 ng total genomic DNA). The horizontal line at 80 in the graph represents three standard deviations from background.

Carry out the preferred embodiment of the present invention:

Unless otherwise required by context, singular terms shall include plural terms and plural terms shall include singular terms.

Unless otherwise indicated, the following terms are consistent with those used in this specification and should be understood as having the following meanings:

A "nucleic acid sequence", "nucleic acid molecule", or "nucleic acid" as used herein refers to one or more oligonucleotides or polynucleotides as defined herein. "Target nucleic acid molecule" or "target nucleic acid sequence" as used herein refers to an oligonucleotide or polynucleotide comprising a sequence in a sample to be detected by a user of the method of the invention.

The term "polynucleotide" herein means a single- or double-stranded nucleic acid polymer of at least 10 bases in length. In certain embodiments, nucleotides, including polynucleotides, may be ribonucleotides or deoxyribonucleotides or modified forms of either. The modification includes base modification such as bromouridine, ribose modification such as arabinoside and 2', 3'-dideoxyribose, and internucleoside bond modification such as phosphorothioate (phosphorothioate), phosphorodithioate ( phosphorodithioate), phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoraniladate, and phosphoroamidate. The term "polynucleotide" especially includes DNA in both single- and double-stranded forms.

Herein the term "oligonucleotide" is meant to include naturally occurring nucleotides, as well as modified nucleotides linked together by naturally and/or non-naturally occurring oligonucleotide bonds. Oligonucleotides are a subset of polynucleotides, comprising members that are usually single stranded and have a length of 200 bases or less. In certain embodiments, the oligonucleotides are 10 to 60 bases in length. In certain embodiments, the oligonucleotides are 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 bases in length. Oligonucleotides may be single-stranded or double-stranded, eg, for the construction of genetic mutants. With respect to sequences encoding proteins, the oligonucleotides of the invention may be sense or antisense oligonucleotides.

The term "naturally occurring nucleotides" includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotide" includes nucleotides or similar nucleotides with modified or substituted sugar groups. The term "oligonucleotide linkage" includes oligonucleotide linkages of phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphordiselenoate, phosphoroanilothioate, phoshoraniladate, phosphoramidate, and the like. See, for example, LaPlanche et al., 1986, Nucl. Acids Res., 14:9081; Stec et al., 1984, J. Am. Chem. Soc., 106:6077; Stein et al., 1988, Nucl. 3209; Zon et al., 1991, Anti-Cancer Drug Design, 6:539; Zon et al., 1991, OLIGONUCLEOTIDES AND ANALOGUES: A PRACTICAL APPROACH, pp.87-108 (F. Eckstein, Ed.), Oxford University Press, Oxford England Uhlmann and Peyman, 1990, Chemical Reviews, 90:543, the disclosures of which are hereby incorporated by reference for any purpose. An oligonucleotide may include a detectable label to enable detection of the oligonucleotide or hybridization thereof.

An "addressable substrate" used in the methods of the invention may be any surface capable of binding oligonucleotides. Such surfaces include, but are not limited to, glass, metal, plastic, or materials coated with functional groups designed to bind oligonucleotides. The coating can be thicker than a monolayer; in fact, the coating can include a porous material thick enough to form a porous three-dimensional structure into which the oligonucleotides can diffuse and bind to their inner surfaces.

The term "capture oligonucleotide" as used herein refers to an oligonucleotide bound to a substrate comprising a nucleic acid sequence that can localize (i.e., hybridize in a sample) a complementary nucleotide sequence or gene to a target nucleic acid molecule, And thus the target nucleic acid molecule is attached to the substrate by the capture oligonucleotide in a hybridized manner. Examples of suitable but non-limiting capture oligonucleotides include DNA, RNA, PNA, LNA, or combinations thereof. Capture oligonucleotides can comprise natural or synthetic sequences, with or without modified nucleotides.

A "detection probe" of the present invention may be any carrier capable of attaching one or more detection oligonucleotides, wherein one or more detection oligonucleotides comprise a nucleotide sequence complementary to a specific nucleic acid sequence. The vector itself may serve as a label, or may comprise or be modified with a detectable label, or the detection oligonucleotide may carry such a label. Carriers suitable for the method of the invention include, but are not limited to, nanoparticles, quantum dots, dendrimers, semiconductors, beads, up-or down-converting phosphors, macromolecular proteins, lipids , carbohydrates, or any suitable inorganic or organic molecule of sufficient size, or combination thereof.

A "detector oligonucleotide" or "detection oligonucleotide" as used herein is an oligonucleotide as defined herein: it comprises a complementary nucleotide sequence or gene localization (i.e. , hybridized in the sample) to the nucleic acid sequence on the target nucleic acid molecule. Suitable but non-limiting examples of detection oligonucleotides include DNA, RNA, PNA, LNA, or combinations thereof. Detecting oligonucleotides may comprise natural or synthetic sequences, with or without modified nucleotides.

The term "label" as used herein refers to a detectable label that can be detected by photonic, electronic, electro-optical, magnetic, gravitational, acoustic, enzymatic, or other physical or chemical means. The term "labeled" refers to the incorporation of a detectable label by, for example, incorporation of a radiolabeled nucleoside or attachment of a detectable label to an oligonucleotide.

As used herein, "sample" refers to any quantity of material comprising nucleic acid that can be used in the methods of the invention. For example, the sample may be a biological sample or may be extracted from a biological sample of human, animal, plant, fungal, yeast, bacterial, viral, tissue or viral origin or a combination thereof. They may comprise or be extracted from solid tissues (eg, bone marrow, lymph nodes, brain, skin), body fluids (eg, serum, blood, urine, saliva, semen, or lymph), skeletal tissue, or individual cells. Alternatively, the sample may comprise purified or partially purified nucleic acid molecules, and eg buffers and/or reagents used to obtain suitable conditions to successfully perform the methods of the invention.

In one embodiment of the present invention, the target nucleic acid molecule in the sample may comprise genomic DNA, genomic RNA, expressed RNA, plasmid DNA, cellular nucleic acid, or nucleic acid derived from cellular organs (such as mitochondria) or parasites, or a combination thereof.

"Biological complexity" of a nucleic acid molecule as used herein refers to the number of nucleotides present in a non-repetitive nucleotide sequence in a nucleic acid molecule, for example, GENE EXPRESSION 2 by Lewin, Second Edition: Eukaryotic Chromosomes, 1980, John Wiley & Sons, New York, which is incorporated herein by reference. For example, a simple oligonucleotide of 30 bases containing non-repetitive sequences has a complexity of 30. The Escherichia coli (E. coli) genome complexity of 4,200,000 base pairs is 4,200,000 because it has essentially no repetitive sequences. However, the human genome has a similar 3,000,000,000 base pairs, many of which are repetitive sequences (eg, approximately 2,000,000,000 base pairs). Similarly the total complexity (ie, number of non-repeating nucleotides) of the human genome is 1,000,000,000.

The complexity of a nucleic acid molecule, such as a DNA molecule, does not depend on the number of distinct repeat sequences (ie, the number of copies of each distinct sequence present in the nucleic acid molecule). For example, if a DNA has 1 sequence of a nucleotides long, 5 copies of a sequence of b nucleotides long, and 50 copies of a sequence of c nucleotides long, its complexity is a+ b+c, the repetition frequency of sequence a is 1, the repetition frequency of sequence b is 5, and the repetition frequency of sequence c is 10.

The total length of different sequences in a given DNA can be determined experimentally by calculating the Cot _1/2 of the DNA, which can be expressed by the following formula,

${\mathrm{<span\; class="notranslate">\; <figure-callout\; id="1"\; label="C\; o\; t"\; filenames="A20038010953300591.png,A20038010953300651.png"\; state="\{\{state\}\}">C</figure-callout></span><figure-callout\; id="1"\; label="C\; o\; t"\; filenames="A20038010953300591.png,A20038010953300651.png"\; state="\{\{state\}\}">\; </figure-callout>}}_{\mathrm{<span\; class="notranslate">\; o</span>}}{}_{}{\mathrm{<span\; class="notranslate">\; t</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}<span\; class="notranslate">\; /</span>\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; =</span>\frac{\mathrm{<span\; class="notranslate">\; 1</span>}}{\mathrm{<span\; class="notranslate">\; k</span>}}$

Among them, C is the concentration of single-stranded DNA at time t _1/2 (when 1/2 of the reaction is completed), and k is the speed constant. Cot _1/2 represents the value required for half-annealing of two complementary strands of DNA. Usually, the renaturation of DNA is represented by a Cot curve, which plots the fraction of DNA remaining single-stranded (C/Co) against the common logarithm (log) of Cot or the renatured DNA Plot of the fraction (1-C/Co) versus the common logarithm of Cot. The Cot curve was proposed by Britten and Kohne in 1968 (1968, Science 161:529-540). The Cot curve shows that the concentration of each renatured sequence determines the renaturation rate of a given DNA. In contrast, Cot _1/2 represents the total length of the different sequences present in the reaction.

The Cot _1/2 of DNA is proportional to its complexity. Therefore, determination of DNA complexity can be accomplished by comparing its Cot _1/2 to the Cot _1/2 of a standard DNA of known complexity. Typically, the standard DNA used to determine DNA complexity is E. coli DNA, which has a complexity consistent with the length of its genome (4.2 × ¹⁰⁶ base pairs), because each sequence in the E. coli genome is considered unique of. Therefore, the following formula can be used to determine the biological complexity of DNA.

In certain embodiments, the present invention provides methods for reliably detecting and distinguishing (i.e., identifying) target nucleic acid molecules with nucleotide mutations (e.g., single nucleotide polymorphisms) in total human DNA, the method It is not necessary to first amplify specific DNA sequences by PCR or any other method for enzymatic complexity reduction. Specifically, the method of the present invention includes a combination of hybridization conditions (including reaction volume, salt, formamide, temperature, and assay mode), a capture oligonucleotide sequence bound to a substrate, a detection probe, and a sufficiently sensitive target nucleic acid molecule Detection means in which target nucleic acid molecules are recognized by both capture oligonucleotides and detection probes.

As shown in the Examples, the present invention provides for the first time a method for the successful detection of SNPs in total human DNA by a one-step hybridization method that does not require upfront amplification or complexity reduction for selective enrichment The target sequence, also without the aid of any enzymatic reaction, involves two hybridization events: the hybridization of a first part of the target sequence to the capture probe, and the hybridization of the second part of the target sequence to the detection probe. Figure 1 shows a schematic diagram of the one-step hybridization method. As discussed above, both hybridization events occur in the same reaction. The target can first bind to the capture oligonucleotide and then hybridize to the detection probe such as the nanoparticle shown in the schematic, or the target can first bind to the detection probe and then hybridize to the capture oligonucleotide.

In another embodiment, the present invention provides methods for reliably detecting and distinguishing (i.e., identifying) target nucleic acid molecules having one or more discrete nucleoside mutations in total DNA without the need to first pass PCR or any other The method preferably amplifies specific DNA sequences for enzymatic complexity reduction. For example, the methods of the invention can be used to distinguish between two or more target nucleic acid molecules from two or more different species of the same genus, where the species differ by two or more discrete nucleotides, The distinction can be made using capture oligonucleotides that differ by one or more nucleotides and/or probe oligonucleotides that differ by one or more nucleotides. The methods of the invention can also be used to distinguish between two or more species within the same genus that differ by two or more consecutive nucleotides.

In one embodiment, the methods of the invention can be practiced using a two-step hybridization method. Figure 2 shows a schematic diagram of the two-step hybridization method. In this method, hybridization events occur in two separate reactions. The target is first bound to the capture oligonucleotide, and after all non-binding nucleic acid is removed, a second hybridization is performed, which provides a detection probe that can specifically bind to a second portion of the captured target nucleic acid.

The method of the present invention involving a two-step hybridization method does not need to confer certain unique adaptive characteristics on the detection probes (such as high Tm and rapid melting behavior) can be implemented because the reaction occurs in two steps. The first step is not rigorous enough to capture only the desired target sequence. Therefore, a second step (binding of the detection probe) is provided to obtain the desired specificity for the target nucleic acid molecule. The combination of these two discriminative hybridization events allows for total specificity for the target nucleic acid molecule. In order to achieve this sharp specificity, however, the hybridization conditions chosen are very stringent. Under such stringent conditions, only a small amount of target and detection probes are captured by the capture probes. The amount of target is usually too small to be detected by standard fluorescence methods because it is buried in the background. Therefore, it is important to the present invention to detect these small numbers of targets using appropriately designed detection probes. The detection probes described herein are present on a carrier portion, usually modified to contain a plurality of detection oligonucleotides, such that the hybridization kinetics of the detection probes are enhanced. Second, the detection probes are also labeled with one or more highly sensitive labeling moieties which, together with appropriate detection means, allow the detection of small numbers of capture target-detection probe complexes. Therefore, it is precisely because of proper adjustment of all factors while using a highly sensitive detection system that the method can be implemented.

The two-step hybridization method of the invention may comprise the use of any of the detection probes described herein in the detection step. In a preferred embodiment, nanoparticle probes are used in the second step of the method. Where and under stringency conditions the nanoparticle probes are used in the second hybridization step as in the first step, the probe oligonucleotides on the nanoparticle probes can be longer than the capture oligonucleotides. In this way, the conditions necessary for the unique characteristics of the nanoparticle probes (high Tm and fast melting behavior) are no longer required.

One-step and two-step hybridization methods, together with appropriately designed capture oligonucleotides and detection probes of the present invention, provide new and unexpected advantages over previous methods for detecting target nucleic acid sequences in samples. In particular, the method of the present invention does not require an amplification step, the purpose of which is to maximize the number of targets in the sample while reducing the relative concentration of non-target sequences to increase the probability of binding to the target, e.g. based on a polymerase Chain reaction (PCR) detection methods require such an amplification step. Specific detection without the need for upfront amplification of the target sequence offers a huge advantage. For example, amplification often leads to contamination of research or diagnostic laboratories, leading to false positive test results. PCR or other targeted amplification requires specially trained personnel, expensive enzymes and specialized equipment. Most importantly, the efficiency of amplification will vary with each target sequence and primer pair, leading to errors or even failures in determining the target sequence or the relative amount of target sequence present in the genome. In addition, the methods of the present invention involve fewer steps and are thus easier to implement and more efficient than gel-based nucleic acid target detection methods, such as Southern and Northern blot assays.

In one embodiment, the present invention provides a method for detecting a target nucleic acid sequence in a sample, wherein the sample comprises a nucleic acid molecule having a biological complexity greater than that of the amplified nucleic acid molecule, the target nucleic acid sequence being identical to a known nucleic acid sequence There is at least one nucleotide difference, the method comprising the steps of: a) providing an addressable substrate bound to a capture oligonucleotide, wherein the capture oligonucleotide can recognize at least a portion of the first portion of the target nucleic acid sequence; b) A detection probe comprising a detection oligonucleotide is provided, wherein the detection oligonucleotide can hybridize with at least a part of the second part of the target nucleic acid sequence in step (a); c) after the capture oligonucleotide can specifically, contacting the sample with the substrate and the detection probe under conditions operative to hybridize selectively to the first portion of the target nucleic acid sequence and the detection probe to specifically and selectively hybridize to the second portion of the target nucleic acid sequence; and d ) detecting whether the capture oligonucleotide and the detection probe hybridize to the first portion and the second portion of the target nucleic acid sequence. In another embodiment, the addressable substrate is bound to a plurality of capture oligonucleotides capable of recognizing portions of a target nucleic acid sequence, and one or more detection probes comprising detection oligonucleotides are capable of interacting with the target nucleic acid sequence. One or more parts of the hybridization, can not be recognized by the capture oligonucleotide.

In another embodiment, the present invention provides a method of identifying a single nucleotide polymorphism in a sample, wherein the sample comprises a nucleic acid molecule of greater biological complexity than the biological complexity of the amplified nucleic acid molecule. The method comprises the steps of: a) providing an addressable substrate bound to at least one capture oligonucleotide, wherein said at least one capture oligonucleotide is capable of recognizing a nucleic acid target comprising a specific polymorphism; b) providing a binding There is a detection probe of a detection oligonucleotide, wherein the detection oligonucleotide is capable of hybridizing to at least a portion of the nucleic acid target of step (a); c) after the capture oligonucleotide is capable of specifically and selectively hybridizing to the nucleic acid target Under the condition that the object is effectively hybridized, and the detection probe can specifically and selectively hybridize effectively with the nucleic acid target, the sample is contacted with the substrate and the detection probe; and d) detecting whether the capture oligonucleotide and the detection probe are compatible with Nucleic acid target hybridization. In another embodiment, the addressable substrate incorporates a plurality of capture oligonucleotides capable of recognizing portions of a target nucleic acid sequence, detection probes comprising detection oligonucleotides are capable of hybridizing to a portion of the target nucleic acid sequence, and Not recognized by capture oligonucleotides.

The method of the invention can distinguish between two sequences that differ by only one nucleotide. Thus, in specific embodiments, the methods of the invention can be used to detect specific target nucleic acid molecules having at least one nucleotide mutation. In preferred embodiments, the mutation is a single nucleotide polymorphism (SNP).

In another embodiment, the detection oligonucleotides can be detectably labeled. Different methods of labeling polynucleotides are known in the art and can be readily applied in the methods described herein. In particular embodiments, the detectable labels of the present invention may be fluorescent, luminescent, Raman-active, phosphorescent, radioactive, or active in scattered light, with unique qualities , or other markers with some other specific detectable physical or chemical properties that are easy to detect, in order to enhance the detectable properties, the markers can be aggregated or one or more copies attached to the carrier, such as dendritic Macromolecules (dendrimers), molecular aggregates, quantum dots or beads. Labeling allows detection by, for example, photonic, electronic, acoustic, photoacoustic, gravitational, electrochemical, enzymatic, chemical, Raman, or mass spectrometric means.

In one embodiment, the detection probes of the invention may be nanoparticle probes bound to detection oligonucleotides. Nanoparticles are objects of great interest due to their unique physicochemical properties derived from their size. Due to these properties, nanoparticles offer a promising avenue for the development of novel biosensors that are more sensitive, specific, and cost-effective than conventional detection methods. The methods for synthesizing nanoparticles and the methodology to study the properties derived from them have been extensively developed in the past 10 years (Klabunde, editor, Nanoscale Materials in Chemistry, Wiley Interscience, 2001). However, due to the inherent incompatibility between these two disparate materials, nanoparticles and biomolecules, there is a lack of robust methods for functionalizing nanoparticles with biomolecules of interest, and thus lead to the development of nanoparticles in biologically significant The application on is limited. Efficient methods for functionalizing nanoparticles with modified oligonucleotides have been developed. See U.S. Patent Nos. 6,361,944 and 6,417,340 (assignee: Nanosphere, Inc.), which are incorporated by reference in their entirety. This method yields nanoparticles highly functionalized by oligonucleotides with surprising particle stability and hybridization properties. The resulting DNA-modified particles are very potent as evidenced by their solution stability, which includes increased electrolytic concentration, stability to centrifugation or freezing, and thermal stability upon repeated heating and cooling. This loading method is controllable and modifiable. Functionalization of nanoparticles of different sizes and compositions, and loading of oligonucleotide recognition sequences onto nanoparticles can be controlled by the loading method. Examples of suitable, but non-limiting nanoparticles include U.S. Patent No. 6,506,564, International Patent Application No. PCT/US02/16382, U.S. Patent Application No. 10/431,341 filed May 7, 2003, and International Patent Application No. Nanoparticles such as those described in Application No. PCT/US03/14100, all of which are hereby incorporated by reference in their entirety.

The aforementioned loading methods for preparing DNA-modified nanoparticles, especially DNA-modified gold nanoparticle probes, have enabled the development of protocols in a new colorimetric sense for oligonucleotides. This method is based on the hybridization of two gold nanoparticle probes to two different regions of a DNA target of interest. Since each probe is functionalized with multiple oligonucleotides of the same sequence, target binding results in the formation of target DNA/gold nanoparticle probe aggregates when sufficient target is present. Recognition of the DNA target results in a colorimetric change due to the shortening of the distance between the particles. This colorimetric change can be monitored optically with a UV-vis spectrophotometer or visually with the naked eye. Additionally, the color deepens as the solution concentrates onto the membrane. Thus, a simple colorimetric change provides evidence of the presence or absence of a specific DNA sequence. Using this test, femtomole and nanomolar concentrations of model DNA targets as well as polymerase chain reaction (PCR) amplified nucleic acid sequences can be detected. Importantly, the gold probe/DNA target complex exhibits a very fast melting transition, making it a highly specific DNA target label. In model systems, insertions, deletions, or mismatches of one base can be easily detected by color and temperature-based spot tests (spot tests) or by spectrophotometrically monitoring the melting transition of aggregates (Storhoff et al., J . Am. Chem. Soc., 120, 1959 (1998)). See also, e.g., U.S. Patent No. 5,506,564.

Due to the sharp melting transition, matches can be detected even in the presence of mismatched targets when hybridization and detection are performed under very stringent conditions (e.g., one degree lower than the melting temperature of a perfectly matched probe/target) perfect target. Hybridization and detection at temperatures close to the melting temperature, along with broader melting transitions such as observed with molecular fluorophore labeling, will result in significant loss of signal due to partial melting of the probe/target complex resulting in a wider melting transition. Low sensitivity, while partial hybridization of mismatched probe/target complexes also leads to low specificity due to the influence of mismatched probe signal. Therefore, nanoparticle probes provide a nucleic acid detection method with higher detection specificity.

As described here, nanoparticle probes, especially gold nanoparticle probes, are surprisingly and unexpectedly suitable for direct SNP detection of genomic DNA without the need for amplification. First, the very rapid melting transition observed in nanoparticle oligonucleotide detection probes translates into unprecedented and surprising detection specificity, allowing single-base discrimination even in the context of the human genome. Second, in DNA microarray-based assays, the silver-based signal amplification method can further provide an ultra-high sensitivity boost.

Nanoparticles may be detected in the methods of the invention using, for example, optical or flatbed scanners. The scanner can be connected to a computer, and the computer is loaded with software capable of calculating the gray value, and the calculated gray value provides a quantitative value of the detected nucleic acid amount.

Appropriate scanners include those used to scan documents into a computer, capable of operating in reflective mode (e.g. flatbed scanners), other devices capable of performing this function or using the same optics, any type of Sensitivity assay devices, and standard scanners adapted for scanning substrates of the invention (eg, flatbed scanners adapted to include a substrate support) (currently, no scanners have been found that can be used in transport mode). The resolution of the scanner must be large enough that the reaction area on the substrate is larger than a single pixel of the scanner. Under the premise that the detectable change produced by the test can be observed relative to the substrate (for example, gray spots produced by silver staining can be observed on a white background, but not on a gray background), the scanner can Use with any substrate. The scanner can be a black and white scanner or preferably a color scanner.

Most preferably, the scanner is a standard color scanner type used to scan documents into a computer. Such scanners are inexpensive and commercially readily available. For example, EpsonExpression 636 (600×600dpi), UMAX Astra 1200 (300×300dpi), or Microtec 1600 (1600×1600dpi) can be used. The scanner is connected to a computer that contains software for processing the images obtained from the scanned substrate. This software can be standard software readily available commercially, such as Adobe Photoshop 5.2 and Corel Photopaint 8.0. Calculation of gray values using these software provides a means of quantifying test results.

These software can also add color numbers to the colored spots, and can produce scanned images (eg, printouts) that can be viewed to obtain qualitative measurements of the presence of nucleic acids, the amount of nucleic acids, or both. It was also found that the sensitivity of the test could be increased by subtracting the color representing a negative result from the color representing a positive result.

The computer may be a standard personal computer, which is readily available commercially. Thus, the use of a standard scanner connected to a standard computer equipped with standard software provides a convenient, easy and inexpensive means of detecting and quantifying nucleic acids when testing is performed on a substrate. Scans (images) can also be stored in a computer, keeping a record of the results for further reference or use. Of course, more complex devices and software could be used if desired.

Silver staining can be used with any type of nanoparticles that catalyze silver reduction. Nanoparticles made of noble metals (eg, gold and silver) are preferred. See Bassell et al., J. Cell Biol., 126, 863-876 (1994); Braun-Howland et al., Biotechniques, 13, 928-931 (1992). If the nanoparticles used for nucleic acid detection do not catalyze the reduction of silver, then silver ions can be bound to nucleic acids to catalyze the reduction. See Braun et al., Nature, 391, 775 (1998). Also, silver-stained regions are known to react with phosphate groups on nucleic acids.

Silver staining can be used to create or enhance detectable changes in any test performed on a substrate, including those described above. In particular, silver staining has been found to greatly increase the sensitivity of assays using a single type of nanoparticle, such that the nanoparticle layer, polymeric probe layer, and core probe layer can often be omitted.

In another embodiment, the oligonucleotide attached to the substrate can be placed between two electrodes, the nanoparticles can be made of a conductive material, and step (d) of the method of the invention can include detecting a change in conductivity. In another embodiment, a number of oligonucleotides in a spotted array are attached to a substrate, each of which can recognize a different target nucleic acid sequence, and each oligonucleotide spot is located between two electrodes In the meantime, the nanoparticles are made of conductive materials, and step (d) of the method of the present invention includes detecting changes in conductivity. The electrodes can be made of gold, for example, and the nanoparticles are also made of gold. Optionally, the substrate can be contacted with silver stained areas to create a change in conductivity.

In specific embodiments, the nucleic acid molecules in the sample have a higher biological complexity than the amplified nucleic acid molecules. Those skilled in the art can readily determine the biological complexity of a target nucleic acid sequence using methods as described in Lewin, GENE EXPRESSION 2, Second Edition: Eukaryotic Chromosomes, 1980, John Wiley & Sons, New York, incorporated herein by reference. Reference.

Hybridization kinetics are absolutely dependent on the concentrations of reaction partners, ie, the strands that must be hybridized. In a given amount of DNA extracted from a cell sample, the amount of DNA for the total genome, mitochondria (if present), and extrachromosomal components (if present) is only a few micrograms. Therefore, the actual concentration of co-reactants to be hybridized will depend on the size of these co-reactants and the complexity of the extracted DNA. For example, when comparing DNA samples of different complexity from different sources, there is one copy of a 30-base target sequence in each genome at different concentrations. For example, the same target sequence is approximately 1,000-fold less concentrated in 1 microgram of total human DNA than in 1 microgram of bacterial DNA and approximately 1,000,000-fold lower than the amount present in 1 microgram of a small plasmid DNA sample.

The high complexity of the human genome (1 x ¹⁰⁹ nucleotides) requires exceptionally high specificity due to redundant and similar sequences in genomic DNA. For example, to distinguish a capture strand with a 25-mer oligonucleotide from the entire human genome, a degree of specificity with a discrimination power of 40,000,000:1 is required. In addition, since wild-type and mutant targets differ by only one base in the 25-mer capture sequence, successful genotyping requires distinguishing between two targets with 96% homology . The present invention surprisingly and unexpectedly provides methods for the efficient, specific and sensitive detection of target nucleic acid molecules of higher complexity compared to amplified nucleic acid molecules.

The biological complexity of target nucleic acid molecules in samples derived from human tissue is 1,000,000,000, but may be 10-fold higher or lower than that of plant or animal genomes. Preferably, the biological complexity is about 50,000 to 5,000,000,000. Most preferably, the biological complexity is about 1,000,000,000.

In one embodiment, the hybridization conditions are effective for specific, selective hybridization of the capture oligonucleotide and/or probe oligonucleotide to the target nucleic acid sequence, under such hybridization conditions, a single base can be detected mismatches, even where the target nucleic acid is part of a nucleic acid sample having a biological complexity of 50,000 or more, such as shown in the Examples below.

The methods of the invention can further be used to identify specific biological microbial species (such as staphylococci) and/or to detect genes that confer antibiotic resistance (for example, the mec A gene that confers resistance to the antibiotic methicillin).

Methicillin-resistant Staphylococcus aureus strains (MRSA) have become the number one nosocomial pathogen worldwide. More than 40% of all hospital-sourced staphylococcal infections in large US teaching hospitals are associated with these bacteria. More recently they have become prevalent in smaller hospitals (20% incidence in hospitals with 200 to 500 beds), and in nursing homes (Wenzel et al., 1992, Am. J. Med. 91 (Supp 3B) :221-7). An unusual and most unfortunate property of MRSA strains is their ability to acquire additional resistance factors that dampen the susceptibility of these strains to other antibiotics used in chemotherapy. Such multi-resistant strains are now endemic worldwide, and the most "advanced" forms of this pathogen carry resistance mechanisms to most available antimicrobial agents (Blumberg et al., 1991, J.Inf.Disease, Vol.63 , pp. 1279-85).

The main genetic component of methicillin resistance is the so-called mec A gene. This gene was found on a stretch of DNA of unknown, non-staphylococcal origin, likely exogenously acquired by ancestral MRSA cells. The mec A gene encodes a penicillin (penicillin) binding protein (PBP) named PBP2A (Murakami and Tomasz, 1989, J.Bacteriol.Vol.171, pp.874-79), which has a very strong low affinity. It appears that PBP2A is a class of "alternative" cell wall synthases that can take over staphylococci when normal PBP (the normal catalyst for cell wall synthesis) supplementation is completely inactivated by beta-lactam antibiotics in the environment and can no longer function crucial task in cell wall synthesis. The important properties of the mecA gene and its antibiotic resistance phenotype gene product PBP2A were confirmed in earlier transposon inactivation experiments, in which the transposon Tn551 was transferred into the mecA gene. As a result of the experiment, the resistance level decreased significantly from the minimum inhibitory concentration (MIC) value of 1600 μg/ml in the parental bacteria to a low value of about 4 μg/ml in the transposon mutant (Matthews and Tomasz, 1990, Antimicrobial Agents and Chemotherapy , Vol.34, pp.1777-9).

Staphylococcal infections in hospitals are becoming more difficult to treat with the rise of antibiotic-resistant strains, and the number of infections caused by coagulase-negative staph species. Effective treatments for these infections are reduced due to the long time required for many of the tests used to determine species (speciation assays) and antibiotic resistance. With the rapid identification of species and antibiotic resistance status, patients can be scheduled earlier and with less use of broad-spectrum antibiotics. Therefore, there is clearly a need for rapid, highly sensitive, and selective identification and differentiation of staphylococcal species and/or methods of detecting the mec A gene.

In another embodiment, the present invention provides oligonucleotide sequences and their antisense complements for staphylococcal speciation assays and/or detection of the methicillin resistance gene (mec A), as well as nanotechnology using these sequences. Particle-labeled probes, methods, and kits. These designed sequences are highly sensitive and selective for staphylococcal species or the mec A gene, which confers some forms of antibiotic resistance. These sequences can be used for the intended purpose of mec A gene detection or staphylococcal speciation analysis, and can also be used as negative controls for other systems. Currently, probe sets of either Tuf3 and Tuf4, or Tuf5 and Tuf6, together with probe Tuf 2 can be used to distinguish S. aureus from S. epidermidis as shown in the Examples below. Sequence-tagged 16S is used to detect the presence of 16S rRNA or DNA contained in Staphylococcus species. Conventional methods such as standard phosphoramidite chemistry can be used to prepare these sequences as capture probes and/or nanoparticle labeling probes.

In another embodiment of the invention, these sequences can be used in staphylococcal speciation assays and/or mec A detection methods using non-amplified genomic DNA. The mec A gene sequence of the present invention has been used to detect the concentration of double-stranded PCR products as low as 1×10 ^-13 M (100fM, 3×10 ⁶ copies) under the current test conditions and methods, and the total genomic DNA content of ultrasonic degradation As little as 33ng (1×10 ⁷ copies) of the mec A gene in a 50 μl reaction (see FIG. 21 ). Sensitivity and specificity of the Tuf gene sequence for S. aureus and S. epidermidis speciation assays were also examined using PCR-amplified gene products or total bacterial genomic DNA. The lower detection limit currently determined is 1×10 ^-12 M (1 pM, or 3×10 ⁷ copies) of double-stranded PCR product in a 50 μl reaction system, and 150 ng (5×10 ⁷ copies) of sonicated genomic DNA ( See Figure 20). The conditions under which these tests were performed are described below. The method of the present invention surprisingly provides efficient, sensitive and specific detection of different staphylococci using bacterial genomic DNA without upfront complexity reduction or targeted amplification by distinguishing DNA sequences with one or more base differences species method.

In yet another embodiment of the invention, when using PCR amplicons, a second nanoparticle probe can be used instead of attaching to the array substrate as described in PCT/US01/46418 (Nanosphere, Inc., Assignee). The captured sequence of the whole is used as a reference. The system can detect by optical means (such as color or light scattering) when target DNA hybridizes to two nanoparticle probes resulting in a color change. This type of test can be used for staph species identification or mec A gene detection purposes as described in the tests above.

Example

The invention will be further illustrated by the following illustrative examples. These examples are provided by way of illustration and are not intended to limit the invention in any way. In these examples, all percentages are by weight if solid and by volume if liquid, and all temperatures are in degrees Celsius unless otherwise indicated.

Example 1

One-step and two-step hybridization methods for SNP identification in non-amplified genomic DNA using nanoparticle probes

Prepared by the methods described in PCT/US97/12783, filed July 21, 1997, PCT/US00/17507, filed June 26, 2000, PCT/US01/01190, filed January 12, 2001 Gold nanoparticle oligonucleotide probes for the detection of target Factor II, MTHFR and Factor V sequences, which are incorporated by reference in their entirety. Figure 3 conceptually illustrates the use of oligonucleotide-conjugated gold nanoparticle probes for the detection of target DNA using DNA microarrays with wild-type or mutant capture probe oligonucleotides. The oligonucleotide sequence bound to the nanoparticle is complementary to one part of the target sequence, and the capture oligonucleotide sequence bound to the glass chip is complementary to another part of the target sequence. Under hybridization conditions, the nanoparticle probe, capture probe, and target sequence bind together to form a complex. The detection signal of the resulting complex can be enhanced with conventional silver staining.

(a) Preparation of gold nanoparticles

Gold colloids (diameter 13 nm) were prepared by reduction of _HAuCl4 with citrate as described by Frens, 1973, Nature Phys. Sci., 241: 20 and Grabar, 1995, Anal. Chem. 67: 735. Briefly, all glassware was cleaned in aqua regia (3 parts HCl, 1 part HNO3), rinsed with Nanopure _H2O , and oven dried before use. _HAuCl4 and sodium citrate were purchased from Aldrich Chemical Company. Hydrated _HAuCl4 (1 mM, 500 mL) was used to stir at reflux. Then, 38.8 mM sodium citrate (50 mL) was added rapidly. The color of the solution changed from pale yellow to burgundy and the reflux was continued for 15 minutes. After cooling to room temperature, filter the red solution using a MicronSeparations Inc. 1 micron filter. Gold colloids were characterized by ultraviolet-visible (UV-vis) spectroscopy using a Hewlett Packard Model 8452A diode array spectrophotometer and by transmission electron microscopy (TEM) using a Hitachi Model 8100 transmission electron microscope. Gold particles with a diameter of 15 nm produce a visible color change when aggregated with target and probe oligonucleotide sequences ranging from 10-35 nucleotides.

(b) Oligonucleotide Synthesis

Using ABI 8909 DNA synthesizer, in single column mode, using phosphoramidite chemistry [Eckstein, F. (ed.) Oligonucleotides and Analogues: A Practical Approach (IRL Press, Oxford, 1991)], synthesize 1 micromolar amount of Capture probe oligonucleotides complementary to fragments of MTHFR, Factor II or Factor V DNA sequences. The capture sequence contains any 3'-amino modifiers that serve as covalently attached reactive groups during array processing. Oligonucleotides were synthesized by the standard DNA synthesis protocol described below. Columns with 3'-amino modifiers attached to solid supports, standard nucleotide phosphoramidites and reagents were obtained from Glen Research. To aid purification, the final dimethoxytrityl (DMT) protecting group was not cleaved from the oligonucleotide. After synthesis, the DNA is cleaved from the solid support using ammonia, resulting in a DNA molecule containing a free amine at the 3' end. With 0.03M Et ₃ NH ⁺ OAc ^- buffer (TEAA), pH7, 95% CH ₃ CN/5% TEAA with a gradient of 1%/min, use a reverse phase column (porous layer solid pellet (Vydac)) Agilent 1100 series equipment was used for reverse phase HPLC chromatography. Flow rate 1mL/min, UV detection at 260nm. After the buffer solution was collected and evaporated, the DMT was cleaved from the oligonucleotide by treating with 80% acetic acid for 30 min at room temperature. The solution was then evaporated to near dryness, water was added, and the cleaved DMT was extracted from the aqueous oligonucleotide using ethyl acetate. The amount of oligonucleotide was determined at absorbance at 260 nm and the final purity was assessed by analytical reverse phase HPLC.

The capture sequences used in testing the MTHFR gene were as follows: MTHFR wild type, 5' GATGAAATCG G CTCCCGCAGAC-NH ₂ 3'(MTHFR-SNP/Cap6-WT22; SEQ ID NO: 1), and MTHFR mutant, 5' ATGAAATCG A CTCCCGCAGACA- NH ₂ 3'(MTHFR-SNP/Cap7-mut22; SEQ ID NO: 2). The capture oligonucleotides corresponding to the Factor V gene are as follows: Factor V wild type, 5' TGG ACA GGC GAG GAA TAC AGGTAT-NH ₂ 3'(FV-Cap-WT24; SEQ ID NO: 3), and Factor V Mutant, 5' CTG GACAGG CAA GGA ATA CAG GTA TT-NH ₂ 3'(FV-Cap-mut26; SEQ ID NO: 4). Factor II wild type: 5'CTCAGCGAGCCTCAATGCTCCC- _NH23 '(FII-SNP/Cap1-WT22; SEQ ID NO: 5), and factor II mutant, 5'CTCTCAGCAAGCCTCAATGCTCC- _NH23 ' (FII-SNP/Cap1- mut23; SEQ ID NO: 6).

Probe probe oligonucleotides designed to detect Factor II, MTHFR and Factor V genes contain a steroidal disulfide bond and are 5' terminated with a recognition sequence. The sequence of the probe is as follows: F II probe, 5'Epi-TCCTGG AAC CAA TCC CGT GAA AGA ATT ATT TTT GTG TTT CTA AAA CT3' (FII-Pro I-47; SEQ ID NO: 7), MTHFR probe, 5′Epi-AAA GAT CCC GGGGAC GAT GGG GCA AGT GAT GCC CAT GTC GGT GCA TGC CTT CACAAA G 3′ (MTHFR-Pro II-58; SEQ ID NO: 8), Factor V probe, 5′Epi-CCA CAGAAA ATG ATG CCC AGT GCT TAA CAA GAC CAT ACT ACA GTG A 3′ (FV-Pro 46; SEQ ID NO: 9).

Probe oligonucleotides were synthesized according to the capture probe synthesis method described below with modifications. First, the amino modifier column was replaced by a support with the appropriate nucleotide representing the 3' end recognition sequence. Second, a steroid-cyclic disulfide at the 5' end was introduced into the ligation step using a modified phosphoramidite containing a steroid disulfide (see Letsinger et al., 2000, Bioconjugate Chem . 11 :289-291 and PCT/US01/01190 (Nanosphere, Inc.), the contents of which are incorporated by reference in their entirety). Phosphoramidites can be prepared by mixing epiandrosterone (0.5 g), 1,2-dithiane-4,5-diol (0.28 g), and toluene (30 mL) under dehydrating conditions. ) in p-toluenesulfonic acid (15 mg) was refluxed for 7 hours (Dean Starkapparatus); the toluene was then removed under reduced pressure and the residue was taken up in ethyl acetate. The solution was washed with water, dried over Na2SO4, concentrated to a syrupy residue which was allowed to stand overnight in pentane/ether to give a white solid with an Rf value (TLC, silicon wafer, ether as extract) of 0.5. Steroid dithioketal (steroid-dithioketal) compound (400mg); In contrast, the Rf values of epiandrosterone and 1,2-dithiane-4,5-diol obtained under the same conditions were 0.4 and 5-diol respectively. 0.3. Recrystallization from pentane/ether gave white powder, mp 110-112°C, ¹ H NMR, δ 3.6 (1H, C ³ OH), 3.54-3.39 (2H, dithiane ring of m 2OCH), 3.2 -3.0 (4H, m 2CH ₂ S), 2.1-0.7 (29H, m steroid H); Mass Spectrum (ES ⁺ ) calcd. for C ₂₃ H ₃₆ O ₃ S ₂ (M+H) 425.2179, found 425.2151 .Anal. (C ₂₃ H ₃₇ O ₃ S ₂ )S: Calculated 15.12; Found 15.26. To prepare steroid-disulfide ketal phosphoramidite derivatives, steroid dithioketal (100 mg) was dissolved in THF (3 mL), cooled in a dry ice alcohol bath, and N was added continuously, N-diisopropylethylamine (80 μL) and β-cyanoethylchlorodiisopropylphosphoramidite (β-cyanoethylchlorodiisopropylphosphoramidite) (80 μL); then, the mixture was heated to room temperature, stirred for 2 hours, and ethyl acetate (100 mL), washed with 5% hydrated NaHCO ₃ and water, dried over sodium sulfate, concentrated to dryness. The residue was placed in a minimum amount of dichloromethane, precipitated by adding hexane at -70°C, and dried in vacuo; product 100 mg; ³¹ P NMR 146.02. After the DNA synthesis was completed, the oligonucleotide linked to epiandrosterone-disulfide was deprotected from the support under ammonia conditions, and purified on HPLC using a reversed-phase column according to the above method.

(c) Oligonucleotides attached to gold nanoparticles

Probes were prepared by first incubating 4 μM oligonucleotide solution and ~14 nM 15 nm citrate-stabilized gold nanoparticle colloidal solution in a final volume of 2 mL for 24 hours. The salt concentration in the solution was gradually increased to 0.8M over 40 hours at room temperature. The resulting solution was passed through a 0.2 μm cellulose acetate filter and spun at 13,000G for 20 minutes to granulate the nanoparticle probes. After removal of suspended solids, the pellet was resuspended in water. In the final step, the probe solution was pelleted again and resuspended in probe storage buffer (10 mM phos, 100 mM NaCl, 0.01% w/v _NaN3 ). After estimating the concentration based on the absorbance at 520 nm (ε = 2.4×10 ⁸ M-1 cm ^-1 ), the concentration was adjusted to 10 nM.

The following nanoparticle-oligonucleotide conjugates specific for Factor II, MTHFR and Factor V DNA were prepared in this way:

Factor II probe: Gold-S'-5'-[TCC TGG AAC CAA TCC CGT GAA AGA ATTATT TTT GTG TTT CTA AAA CT-3'] _n (FII-ProI-47; SEQ ID NO: 10)

MTHFR probe: Gold-S'-5'-[AAA GAT CCC GGG GAC GAT GGG GCA AGTGAT GCC CAT GTC GGT GCA TGC CTT CAC AAA G-3'] _n (MTHFR-II58; SEQ ID NO: 11)

Factor V probe: Gold-S'-5'-[CCA CAG AAA ATG ATG CCC AGT GCT TAACAA GAC CAT ACT ACA GTG A-3'] _n (FV-46; SEQ ID NO: 12)

S' refers to the linker unit prepared through the epiandrosterone disulfide group; n represents the number of recognition oligonucleotides.

(d) Preparation of DNA microarray

Capture strands were arrayed on Superaldehyde slides (Telechem) or CodeLinke slides (Amersham, Inc.) using a GMS417 arrayer (Affymetrix). The arrangement of arrayed spots was designed to allow multiple hybridizations on each slide and was obtained by dividing the slides into individual test wells with silicone spacers (Grace Biolabs). Wild-type and mutant types were spotted three-fold in the spotting buffer provided by the manufacturer. Follow the protocol recommended by the manufacturer for post-processing of slides.

(e) hybridization

Factor V SNP Detection Test Method

Factor V SNP detection was performed using the protocol described below. Sonicated human placental DNA genotyped homozygous wild type, or salmon sperm DNA (Sigma) was ethanol precipitated and dissolved in 10 nM FV probe solution. Other components were added to the mixture, and the final hybridization mixture (5 μL) contained 3xSSC, 0.03% Tween20, 23% formamide, 5 nM FV probe, and 10 μg human DNA, or refer to the instructions. After a 99°C, 4 minute heat denaturation step, the hybridization mixture was added to the detection wells. The arrays were incubated at 50°C for 90 minutes. Washing after hybridization was initiated by immersing the array in 0.5M NaNO ₃ , 0.05% Tween 20 for one minute at room temperature. Remove the spacer, rinse the detection slide again in 0.5M NaNO ₃ /0.05% Tween 20 solution, and incubate at room temperature for 3 minutes (2x), with slight agitation during the incubation. Slides were stained with silver enhancement solution, dried on a spin dryer, and imaged on an Array Worx(R) biochip reader (Model no. AWE, Applied Precision Inc., Issaquah, WA, USA) as described above.

(f) Results

Factor V SNP detection

Figure 4 shows the SNP discrimination of Factor V gene in human genomic DNA on Superaldehyde slides. The test array contained wild-type and mutant capture spots. The array shown on the top was hybridized to wild-type human genomic DNA and the array on the bottom was hybridized to sonicated salmon sperm DNA. The signal of the wild-type puncta was significantly higher than that of the mutant puncta hybridized to wild-type human genomic DNA, indicating a wild genotype homozygous for factor V. Under hybridization conditions, no hybridization signal of salmon sperm DNA was observed, which was used as a test control. SNP calling was also performed using the arrays on CodeLink(R) slides.

Experiments were designed to show that hybridization on wild-type capture plaques is not due to some other sequence, but is specific to genomes containing the human Factor V gene. Using human wild-type total DNA, the expected strong hybridization signal was observed in wild-type capture spots, while an approximately 3-fold weaker signal was observed in mutant spots. However, no signal was observed when genomic DNA extracted from salmon sperm was used as the target because this DNA does not contain the human Factor V gene.

Figure 5 shows the importance of adjusting hybridization conditions in order for this method to discriminate between two target nucleic acids that differ by 1 nucleotide (SNP site), in this case from a patient homozygous for a Factor V gene mutation . An appropriate balance of formamide and SSC buffer salt concentration must be determined to allow the target sequence to preferentially bind its cognate capture probe (eg Mut-A or Mut-B sequence). Additionally, Figure 5 shows the effect of different sizes of capture oligonucleotide sequences on the hybridization. The Mut-A sequence is 26 nucleotides long and the Mut-B sequence is 21 nucleotides long. The results showed a significant difference in the specific signal under the 15% FM/1XSSC condition, but no difference under the 25% FM/6XSSC condition, and both probes produced strong signals with good discrimination.

To determine whether it is possible to detect more than one SNP type in a sample under the same conditions, genomic DNA was tested for the presence of wild-type and mutant Factor II and Factor V genes. Normal human (wt) genomic DNA, capture oligonucleotides attached to the substrate, and nanoparticle probes were mixed together in 35% FM and 4X SSC at 40°C for 1 hour. Signal was preferentially generated in wild-type capture spots of Factor II and Factor V genes (Figure 6). When using total genomic DNA from individuals homozygous for mutant factor II and homozygous wild-type factor V, the same array under the same hybridization conditions preferentially produced signal at the factor II mutant capture spot and factor V wild-type capture spot , clearly and unmistakably identified the genetic composition of this person on the SNP configuration of these two genes (Fig. 6). This result shows that capture oligonucleotide sequences and hybridization conditions can be designed such that more than one SNP type can be detected in the same array under the same hybridization conditions. Also, SNP discrimination between wild-type and mutant DNA is possible independent of whether the input DNA is of normal or mutant origin.

two-step hybridization

To determine the effect of different stringency conditions on SNP discrimination, more experiments were performed. The detection arrays were hybridized under different stringency conditions using different percentages of formamide in the assay (Figure 7). With increasing stringency, there is a loss of signal, which in turn increases the specificity of the signal. Little signal was observed in the no target control. Quantification of the signal in assay spots showed that the signal was 3-6 times higher for wild-type spots than for mutant spots (Fig. 7B). These results also provide support for the identification of SNPs in genomic DNA without requiring any targeted amplification strategy.

The different lengths include 20, 21, 24, or 26 nucleotides (FV-WT20 (SEQ ID NO: 13): 5'(GGACAGGCGAGGAATACAGG)-(PEG)x3-NH ₂ , 3'FV-mut21 (SEQ ID NO :14): 5'(TGGACAGGCAAGGAATACAGG)-(PEG)x3-NH ₂ 3', FV-wt24 (SEQ ID NO: 15): 5'TGG ACA GGC GAG GAA TAC AGG TAT-NH ₂ 3', FV-mut26 (SEQ ID NO: 16): 5′ CTG GAC AGG CAA GGA ATA CAG GTATT-NH ₂ 3′) capture oligonucleotides were printed on CodeLink slides as described above, and added to 5 μg of normal human placenta genomic DNA (Sigma, St. Louis, MO) or Factor V mutant human genomic DNA (isolated from repository culture GM14899, Factor V deficient, Coriell Institute). In the first step, slides and DNA were placed in 20% FM, 30% FM or 40% FM, and 4X SSC/0/04% Tween at 40°C for 2 hours. Slides were then rinsed in 2XSSC for 3 minutes at room temperature. After washing, the probe oligonucleotides recognizing Factor V were added together with the nanoparticle probes, and the mixture was incubated at 40° C. for 1 hour. Signals were detected by silver staining as described above. The results show that under optimally adjusted conditions (30% FM in this case), human wild-type DNA only produces a signal on the wild-type probe, whereas human mutant DNA produces a signal only on the mutation-capture probe (Fig. 8). Changing the stringency of the conditions results in loss of discrimination (too low stringency) or loss of signal (too high stringency). Figure 9 shows quantitative data for preferred (intermediate) hybridization conditions in Figure 8 .

The experiment was then repeated under optimal conditions using different concentrations of DNA. As seen in Figure 10, SNP discrimination was successful at DNA concentrations of 0.5 μg, 1.0 μg and 2.5 μg. Therefore, this method is capable of detecting SNPs with very small amounts (less than 1 microgram) of total human DNA. These results also show the importance of capture oligonucleotide design, and stringency conditions properly matched to the length and nucleoside composition of the capture (and detection) probes.

The reproducibility of the two-step hybridization method was determined by performing 10 identical hybridizations with 5 μg of wild-type whole genomic DNA in different wells of a slide. As shown in Figure 11, the standard deviations of net signal intensities for matches and mismatches in 10 individual hybridizations did not overlap, showing that for each hybridization reaction, the SNP configuration of the input DNA could be reliably determined.

Next, this method was used to detect SNPs for Factor V, Factor II, and MTHFR and to prepare wild-type genes in the same samples. Capture oligonucleotides for Factor V, Factor II, or MTHFR were incubated with 5 μg of intact genomic DNA under the hybridization conditions described above. In the second step, nanoparticle probes specifically designed to detect Factor V, Factor II, or MTHFR are added. The results of this experiment are shown in Figures 12-14, which show that under the same conditions, the SNP configurations of at least 3 different genes can be analyzed simultaneously in a single array. Figure 13 shows the results of this multiplex SNP detection in a patient DNA sample (GM16028), which was heterozygous for each gene. Figure 14 shows the results of multiple SNP detection in a patient who is heterozygous for Factor II, wild-type for Factor V, and mutated for MTHFR. This method accurately identified the genotype of this patient (patient sample GM00037). These results show that the discriminative power is strong enough to discriminate between homozygous and heterozygous mutant genes. For example, for any given SNP, a person can be homozygous wild type, mutant, or heterozygous (ie, one wild type gene and one mutant gene). These three different situations for the three separate SNP sites can be correctly identified separately in a single test. The results show that the method of the present invention is capable of simultaneously identifying multiple SNPs in a single sample. While only three SNPs were identified in this assay, those skilled in the art will recognize that this is only a representative number. It is possible to detect more SNP sites in the same array.

In addition to these experiments, two different experimenters also hybridized 8 different slides with DNA from 2 different patients (1 slide per slide for patient GM14899) using the methods described in these examples. arrays, DNA from patient GM1600 is 2 arrays per slide). There were 4 replicate spots per array for each of the 2 genes (factor II and factor V) and each type of capture probe (mutant or wild type). The net signal strengths are averaged, sorted, and plotted starting with the lowest signal strength. For mismatch signals (lower values in each graph), the standard deviation was added to the mean net signal in triplicate. The signals of the mutant and the corresponding wild type are always on top of each other. As shown in Figure 15, even at the smallest signal strengths, the matched net signal was always greater than the mismatched net signal plus three times the standard deviation. Thus, in each instance, the correct configuration of the SNP could be determined with greater than 99% reliability. These results further demonstrate the robustness of the method described here and its reproducibility.

Example 2

Hybridization conditions of the method of the present invention

The standard recommended protocol (T. Maniatis, EFFritsch, and J. Sambrook in "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory, 1982, p324) described in the prior art for efficient hybridization reactions mainly stipulates that the hybridization temperature is low At Tm ~ 10-20 degrees Celsius, this Tm is calculated according to the selected hybridization conditions, including salt and formamide concentration. There are different methods for calculating Tm values, each based on the exact oligonucleotide sequence and buffer conditions. For example, such calculations can be performed using computer programs, commercially available or on-line, such as the HYTHER( ^TM) server developed at the Wayne State University website. The inventors used all available programs on the HYTHER ^™ server to perform these calculations, resulting in Tm values for capture and detection probes (ie oligonucleotides). As shown in Table 1, the Tm values of the capture probes were lower than or very close to the temperature selected for hybridization (ie, 40°C). Therefore, under these conditions, hybridization efficiency can be expected to be very low. In addition, the capture oligonucleotides are directly attached to the substrate surface, i.e., without linker sequences, which means that the oligonucleotides closest to the surface may not be able to participate in the hybridization with the target sequence, thus further reducing the effective Tm value. Based on the teachings of the prior art, the conditions used in the method of the present invention unexpectedly achieve highly efficient hybridization, especially when the target sequence is only a very small fragment (such as 1/100,000,000 or 1 million) of the complex DNA mixture represented by the human genome. 's%) case.

Table 1 TM calculated with HyTher ^TM (Wayne State University) no correction TM correction for hybridization to surface-bound probes as follows (35%FM) Santalucia et al. (35% FM) Fotin et al. (35% FM) catch sequence FII-SNP/Cap1-wt22 (SEQ ID NO: 5) CTCAGC G AGCCTCAATGCTCCC 46.7 37.0 45.0 FII-SNP/Cap1-mut23 CTCTCAGC A AGCCTCAATGCTCC 47.2 35.7 46.3 MTHFR-SNP/Cap6-wt22 (SEQ ID NO: 6) GATGAAATCG G CTCCCGCAGAC 40.3 35.5 43.0 MTHFR-SNP/Cap7-wt22 (SEQ ID NO: 2) ATGAAATCG A CTCCCGCAGACA 40.7 36.2 44.0 FV-Cap-WT-24 (SEQ ID NO: 15) TGGACAGGCGAGGAATACAGGTAT 44.8 35.5 42.9 FV-Cap-mut26 (SEQ ID NO: 16) CTGGACAGGCAAGGAATACAGGTATT 44.5 35.8 42.9 probe FV-46 (SEQ ID NO: 12) 5'Epi-CCA CAG AAA ATG ATGCCC AGT GCT TAA CAA GAC CATACT ACA GTG A 3' 54.8 49.2 57.6 FII-ProI-47 (SEQ ID NO: 10) 5'Epi-TCC TGG AAC CAA TCCCGT GAA AGA ATT ATT TTT GTGTTT CTA AAA CT 3' 52.2 46.9 54.8 MTHFR-Pro II-58 (SEQ ID NO: 8) 5'Epi-AAA GAT CCC GGG GACGAT GGG GCA AGT GAT GCCCAT GTC GGT GCA TGC CTT CACAAA G 3' 68.4 58.6 68.5

Example 3

Preparation of Nanoparticle-Oligonucleotide Conjugated Probes

In this example, representative nanoparticle-oligonucleotide conjugated probes for PCR amplification of mec A and Tuf gene targets were prepared. Using the method described in PCT/US97/12783, filed July 21, 1997, PCT/US00/17507, filed June 26, 2000, PCT/US01/01190, filed January 12, 2001 Preparation of gold nanoparticles-oligonucleotide probes for detection of target mec A or Tuf gene sequences, the entirety of these documents is incorporated by reference.

(a) Preparation of gold nanoparticles

Gold colloids (13 nm in diameter) were prepared by reduction of _HAuCl4 with citrate as described by Frens, 1973, Nature Phys. Sci., 241 : 20 and Grabar, 1995, Anal. Chem. 67 : 735. Briefly, all glassware was cleaned in aqua regia (3 parts HCl, 1 part HNO ₃ ), rinsed with Nanopure H ₂ O, and oven dried before use. _HAuCl4 and sodium citrate were purchased from Aldrich Chemical Company. Hydrated _HAuCl4 (1 mM, 500 mL) was used to stir at reflux. Then, 38.8 mM sodium citrate (50 mL) was added rapidly. The color of the solution changed from pale yellow to burgundy and the reflux was continued for 15 minutes. After cooling to room temperature, filter the red solution using a MicronSeparations Inc. 1 micron filter. The gold colloids were characterized by ultraviolet-visible (UV-vis) spectroscopy using a Hewlett Packard Model 8452A diode array spectrophotometer and by transmission electron microscopy (TEM) using a Hitachi Model 8100 transmission electron microscope. Gold particles with a diameter of 13 nm produce a visible color change when aggregated with target and probe oligonucleotide sequences ranging from 10-35 nucleotides.

(b) Synthesis of Steroid Disulfides

Using a Milligene Expedite DNA synthesizer, in single-column mode, using phosphoramidite chemistry [Eckstein, F. (ed.) Oligonucleotides and Analogues: A Practical Approach (IRL Press, Oxford, 1991)], synthesize 1 micromolar Quantities of oligonucleotides complementary to mecA and Tuf DNA sequences. All solutions were purchased from Milligene (DNA synthesis grade). Average ligation efficiencies ranged from 98 to 99.8%, and to aid purification, the final dimethoxytrityl (DMT) protecting group was not cleaved from the oligonucleotide.

To facilitate hybridization of the probe sequence to the target, the 5' end of the probe sequence includes a deoxyadenosine oligonucleotide as a spacer (da ₁₅ peg for all probes except the Tuf2 probe which has da ₁₀ peg) .

For the generation of 5' steroidal cyclic disulfide oligonucleotide derivatives (see Letsinger et al., 2000, Bioconjugate Chem. 11 :289-291 and PCT/US01/01190 (Nanosphere, inc.) its disclosure in its entirety And as a reference), the final ligation reaction was carried out on the automatic synthesizer of Applied Biosystems with cyclic dithiane-linked epiandrosterone phosphoramidite, which was formed from 1 , 2-dithiane-4,5-diol, epiandrosterone and p-toluenesulfonic acid (PTSA) in toluene. The phosphoramidite reagent can be prepared as follows: epiandrosterone (0.5 g), 1,2-dithiane-4,5-diol (0.28 g) and p-toluenesulfonic acid (15 mg ) solution was refluxed for 7 hours under dehydration conditions (Dean Stark apparatus). Then toluene was removed under reduced pressure, and the residue was placed in ethyl acetate. The solution was washed with water, dried over sodium sulfate, concentrated to a syrupy residue, and left overnight in pentane/ether to give steroid dithioketal compound (400 mg) as a white solid; Rf value (TLC, silicon wafer, ether as extract) was 0.5; in contrast, the Rf values of epiandrosterone and 1,2-dithiane-4,5-diol obtained under the same conditions were 0.4 and 0.3, respectively. Recrystallization from pentane/ether gave a white powder, mp 110-112°C, ¹ H NMR, δ 3.6 (1H, C ³ OH), 3.54-3.39 (2H, dithiane ring of m2OCH), 3.2- 3.0 (4H, m 2CH ₂ S), 2.1-0.7 (29H, m steroid H); Mass Spec (ES ⁺ ) calculated for C ₂₃ H ₃₆ O ₃ S ₂ (M+H) 425.2179, found 425.2151. Anal. ( _{C23H37O3S2 )} S: _{Calculated 15.12} ; Found 15.26. To prepare steroid disulfide ketone phosphoramidite derivatives, steroid dithioketal (100 mg) was dissolved in THF (3 mL), cooled in a dry ice alcohol bath, and N,N-diisopropylethylamine ( 80 μL) and β-cyanoethyl chloride diisopropyl phosphoramidite (80 μL); then, the mixture was warmed to room temperature, stirred for 2 hours, mixed with ethyl acetate (100 mL), washed with 5% hydrated NaHCO ₃ and water , dried over sodium sulfate, concentrated to dryness. The residue was placed in a minimum amount of dichloromethane, precipitated by adding hexane at -70°C, and dried in vacuo; product 100 mg; ³¹ P NMR 146.02. Epiandrosterone disulfide-linked oligonucleotides were synthesized on an Applied Biosystems automated gene synthesizer without removing the final DMT. Upon completion, epiandrosterone disulfide-linked oligonucleotides were deprotected from the support under ammonia conditions and purified on HPLC using a reverse-phase column.

Use 0.03M Et ₃ NH ⁺ OAc ^- buffer (TEAA), pH 7, 1%/min gradient of 95% CH ₃ CN/5% TEAA, on a Hewlett Packard ODS hypersil column (4.6×200mm, particle size of 5 mm) on a Dionex DX500 system for reverse phase HPLC. The flow rate was 1 mL/min with UV detection at 260 nm. DMT-protected unmodified oligonucleotides were purified by preparative HPLC. After the buffer was collected and evaporated, DMT was cleaved from the oligonucleotide by treating with 80% acetic acid for 30 min at room temperature. The solution was evaporated to near dryness, water was added, and the cleaved DMT was extracted from the aqueous oligonucleotide solution with ethyl acetate. The amount of oligonucleotide was determined at absorbance at 260 nm and the final purity was assessed by reverse phase HPLC.

(c) Microarray preparation

Synthesize DNA containing 3'-amino and 5'-amino groups on a DNA synthesizer according to the following standard DNA synthesis protocol. Amine-modified DNA was attached to acetaldehyde microarray slides by printing 1 mM DNA solution in ArrayIt buffer plus (Catalog no. MSP, Companyname Telechem, city Sunnyvale, State CA). Microarrays were oriented on slides using an Affymetrix GMS 417 arrayer with 500 micron print pins. Microarray slides with acetaldehyde-functionalized surfaces were purchased from Telechem (catalog no. SMM, city Sunnyvale, state CA). After printing, slides were placed in a humid chamber for 12-18 hours at ambient temperature. The slides were removed and vacuum dried for 30 minutes to 2 hours. Slides were then rinsed twice in 0.2% w/v SDS and twice in water to remove any residual unbound DNA. Slides were then soaked in 2.5M sodium borohydride in 20% v/v 100% ethanol in 1X PBS for 5 minutes. It was then washed three times with 0.2% w/v SDS, twice with water, and dried by centrifugation.

(d) Oligonucleotides attached to gold nanoparticles

The citrate-stabilized gold nanoparticle colloid solution (approximately 10 nM) prepared according to the method described in part A above was mixed with the sulfur-modified-a _{15 peg} -probe oligonucleotide (4 μM) prepared according to the method described in part B, It was left to stand in a 20 ml scintillation vial for 6 hours at room temperature. Both 0.1M sodium hydrogen phosphate buffer at pH 7.0 and 5.0M NaCl were added to the solution to obtain a solution of 0.01M sodium hydrogen phosphate and 0.1M NaCl, which was left to stand for a further 16 hours. Sodium chloride was added in a gradient to bring the NaCl concentration to 0.8 M within 36 hours, and the resulting solution was incubated for an additional 18 hours. The solution was evenly divided into 1ml eppendorf tubes, and centrifuged at 14,000rpm in Eppendorf Centrifuge 5414 for 25 minutes to obtain a very light pink supernatant, which contained most of the oligonucleotides (shown by the absorbance value at 260nm) together with 7-10% colloidal gold (indicated by absorbance value at 520 nm), as a dense, black gelatinous residue at the bottom of the test tube. Remove the supernatant and resuspend the residue in the desired aqueous buffer. In this example, the buffer solution used includes 0.1M NaCl, 10mM sodium citrate and 0.01% sodium azide, and the pH value is 7.

The following nanoparticle-oligonucleotide detection probes and amine-modified DNA capture probes specific for mecA and Tuf DNA were prepared in this way: Here, the oligonucleotide probes can be amine-modified and immobilized on glass On-chip as capture probes, or modified with epiandrosterone linkers and immobilized on gold particles as detection probes. In other words, an oligonucleotide and its reverse complement can be used interchangeably as capture probes or nanoparticle detection probes.

(a) Detection probe

Probe Tuf 1: Gold-S'-5'-[a ₁₅ PEG-ttctatttccgtactactgac-3'] _n (SEQ ID NO: 17)

Probe Tuf 2: Gold-S'-5'-[a ₁₅ peg-ttctatttccgtactactgacgtaact-3'] _n (SEQ ID NO: 18)

Probe Tuf 3: 5'-[amine- _peg3 -ccattcttctcaaactatcgt-3'] (SEQ ID NO: 19)

Probe Tuf 4: 5'-[amine- _peg3 -ccattcttcactaactatcgc-3'] (SEQ ID NO: 20)

Probe Tuf 5: 5'-[amine- _peg3 -cacactccattcttctcaaact-3'] (SEQ ID NO: 21)

Probe Tuf 6: 5'-[amine- _peg3 -cacactccattcttcactaact-3'] (SEQ ID NO: 22)

Probe Tuf 7: 5'-[amine- _peg3 -atatgacttcccaggtgac-3'] (SEQ ID NO: 23)

Probe Tuf 8: 5'-[amine- _peg3 -gtagatacttacattcca-3'] (SEQ ID NO: 24)

Probe Tuf 9: 5'-[amine- _peg3 -gttgatgattacattcca-3'] (SEQ ID NO: 25)

Probe Tuf 10: 5'-[amine- _peg3 -ccattcttcactaactaccgc-3'] (SEQ ID NO: 26)

Probe Tuf 11: 5'-[amine- _peg3 -catacgccattcttcactaact-3'] (SEQ ID NO: 27)

Probe Tuf 15: 5'-[amine- _peg3 -ccattcttctctaactatcgt-3'] (SEQ ID NO: 28)

Probe Tuf 16: 5'-[amine- _peg3 -ccattcttcacaaactatcgt-3'] (SEQ ID NO: 29)

Probe Tuf 17: 5'-[amine- _peg3 -ccattcttcagtaactatcgc-3'] (SEQ ID NO: 30)

Probe Tuf 18: 5'-[amine- _peg3 -ccattcttcagtaactaccgc-3'] (SEQ ID NO: 31)

Probe Tuf 19: 5'-[amine- _peg3 -ccattcttctcaaactaccgc-3'] (SEQ ID NO: 32)

Probe Tuf 20: 5'-[amine- _peg3 -ccattcttctctaactaccgt-3'] (SEQ ID NO: 33)

Probe Tuf 21: 5'-[amine- _peg3 -catacgccattcttcagtaact-3'] (SEQ ID NO: 34)

Probe Tuf 22: 5'-[amine- _peg3 -cacactccattcttcagtaact-3'] (SEQ ID NO: 35)

Probe Tuf 23: 5'-[amine- _peg3 -catactccattcttcactaact-3'] (SEQ ID NO: 36)

Probe Tuf 24: 5'-[amine- _peg3 -catacaccaattcttctcaaact-3'] (SEQ ID NO: 37)

Probe Tuf 25: 5'-[amine- _peg3 -catactccattcttctctaact-3'] (SEQ ID NO: 38)

Probe Tuf 26: 5'-[amine- _peg3 -cacactccattcttcacaaact-3'] (SEQ ID NO: 39)

Probe Tuf 27: 5'-[amine- _peg3 -cacactccattcttctctaact-3'] (SEQ ID NO: 40)

Probe mecA 1: 5'-[amine- _peg3 -tcgatggtaaaggttggc-3'] (SEQ ID NO: 41)

Probe mecA 2: 5'-[amine- _peg3 -atggcatgagtaacgaagaatata-3'] (SEQ ID NO: 42)

Probe mecA 3: Gold-S'-5'-[amine-peg ₃ -aaagaacototgotcaacaag-3']n (SEQ ID NO: 43)

Probe mecA 4: Gold-S'-5'-[amine- _peg3 -gcacttgtaagcacaccttcat-3']n (SEQ ID NO: 44)

Probe mecA 6: 5'-[amine- _peg3 -ttccagattacaacttcacca-3'] (SEQ ID NO: 45)

Probe 16S 12: 5'-[amine- _peg3 -gttcctccatatctctgcg-3'] (SEQ ID NO: 46)

Probe 16S 13: Gold-S'-5'-[amine- _peg3 -atttcacogotacacatg-3']n (SEQ ID NO: 47)

s' denotes linker units prepared via epiandrosterone disulfide groups; n denotes different numbers of oligonucleotides used in the preparation of nanoparticle-oligonucleotide conjugates.

Table 2 name SEQ ID NO : Sequence 5'->3' Staphylococcus species Tuf1 1748 TFCTATTTCCGTACTACTGACGTCAGTAGTACGGAAATAGAA (reverse complement) common Tuf gene Tuf2 1849 TTCTATTTCCGTACTACTGACGTAACTAGTTACGTCAGTAGTACGGAAATAGAA (reverse complement) common Tuf gene Tuf3 1950 CCATTCTTCTCAAACTATCGTACGATAGTTTGAGAAGAATGG (reverse complement) Staphylococcus aureus Tuf4 205l CCATTCTTCACTAACTATCGCGCGATAGTTAGTGAAGAATGG (reverse complement) Staphylococcus epidermidis Tuf5 2l52 CACACTCCATTCTTCTCAAACTAGTTTGAGAAGAATGGAGTGTG (reverse complement) Staphylococcus aureus Tuf6 2253 CACACTCCATTCTTCACTAACTAGTTAGTGAAGAATGGAGTGTG (reverse complement) Staphylococcus epidermidis Tuf7 2354 ATATGACTTCCCAGGTGACGTCACCTGGGAAGTCATAT (reverse complement) common Tuf gene Tuf8 2455 GTAGATACTTACATTCCATGGAATGTAAGTATCTAC (reverse complement) Staphylococcus aureus Tuf9 2556 GTTGATGATTACATTCCATGGAATGTAATCATCAAC (reverse complement) Staphylococcus epidermidis Tuf10 2657 CCATTCTTCACTAACTACCGCGCGGTAGTTAGTGAAGAATGG (reverse complement) Staphylococcus saprophyticus mimicking Staphylococcus (S.simulans) Tuf11 2758 CATACGCCATTCTTCACTAACTAGTTAGTGAAGAATGGCGTATG (reverse complement) Staphylococcus saprophyticus Tuf15 2859 CCATTCTTCTCTAACTATCGTACGATAGTTAGAGAAGAATGG (reverse complement) Staphylococcus hominis Tuf16 2960 CCATTCTTCACAAACTATCGTACGATAGTTTGTGAAGAATGG (reverse complement) Staphylococcus haemolyticus Tuf17 306l CCATTCTTCAGTAACTATCGCGCGATAGTTACTGAAGAATGG (reverse complement) Staphylococcus cochlii (S.cohnii) Tuf18 3162 CCATTCTTCAGTAACTACCGCGCGGTAGTTACTGAAGAATGG (reverse complement) S.warneri Staphylococcus capitis (S.capitis) Tuf19 3263 CCATTCTTCTCAAACTACCGCGCGGTAGTTTGAGAAGAATGG (reverse complement) Staphylococcus Lyonis (S. lugdunensis) Tuf20 3364 CCATTCTTCTCTAACTACCGTACGGTAGTTAGAGAAGAATGG (reverse complement) Staphylococcus auris (S.auricularis) Tuf21 3465 CATACGCCATTCTTCAGTAACTAGTTACTGAAGAATGGCGTATG (reverse complement) Staphylococcus coli Tuf22 3566 CACACTCCATTCTTCAGTAACTAGTTACTGAAGAATGGAGTGTG (reverse complement) S.warneri Staphylococcus capitis Tuf23 3667 CATACTCCATTCTTCACTAACTAGTTAGTGAAGAATGGAGTATG (reverse complement) Mimic Staphylococcus Tuf24 3768 CATACACCATTCTTCTCAAACTAGTTTGAGAAGAATGGTGTATG (reverse complement) Staphylococcus Lyonis Tuf25 3869 CATACTCCATTCTTCTCTAACTAGTTAGAGAAGAATGGAGTATG (reverse complement) Staphylococcus hominis Tuf26 3970 CACACTCCATTCTTCACAAACTAGTTTGTGAAGAATGGAGTGTG (reverse complement) Staphylococcus haemolyticus Tuf27 4071 CACACTCCATTCTTCTCTAACTAGTTAGAGAAGAATGGAGTGTG (reverse complement) Staphylococcus auris MecA1 4172 TCGATGGTAAAGGTTGGCGCCAACCTTTACCATCGA (reverse complement) mecA gene MecA2 4273 ATGGCATGAGTAACGAAGAATATATATTGTATTCGTTACTCATGCCAT (reverse complement) mecA gene MecA3 4374 AAAGAACCTCTGCTCAAACAAGCTTGTTGAGCAGAGGTTCTTT (reverse complement) mecA gene MecA4 4475 GCACTTGTAAGCACACCTTCATATGAAGGTGTGCTTACAAGTGC (reverse complement) mecA gene MecA6 4576 TTCCAGATTACAACTTCACCATGGTGAAGTTGTAATCTGGAA (reverse complement) 16S12 4677 GTTCCTCCATATCTCTGCGCGCAGAGATATGGAGGAAC (reverse complement) 16s rRNA 16S13 4778 ATTTCACCGCTACACATGCATGTGTAGCGGTGAAAT (antisense complement) 16s rRNA

Example 4

Detection of the mec A gene sequence from bacterial genomic DNA using gold nanoparticle probes

In this example, a method for detecting the sequence of the mec A gene using gold nanoparticles based detection in an array layout is described. Microarray plates with mecA 2 and mecA 6 oligonucleotides as capture probes were used together with mecA 4 oligonucleotide detection probe labeled gold nanoparticles. Microarray plates, capture probes and detection probes were prepared according to the method described in Example 3.

Gold nanoparticles (13 nm in diameter) with attached oligonucleotide probes prepared as described in Example 3 were used to reveal the presence of mec A DNA hybridized to a transparent substrate in the form of a three-component sandwich test. Then, nanoparticles with attached probe oligonucleotides and genomic DNA targets isolated from methicillin-resistant (MecA+) or methicillin-sensitive (MecA-) S. aureus bacterial cells were co-hybridized to these substrates superior. Thus, the presence of nanoparticles on the surface indicated the detection of the mec A gene sequence, see FIG. 16 . At the target amounts tested (250 ng (7.5E7 copies) - 1 μg (3.0E8)), attached nanoparticles could not be observed with the naked eye. To facilitate the observation of nanoparticles hybridized on the substrate surface, a signal amplification method was employed in which silver ions were catalytically reduced with hydroquinone to form silver metal on the slide surface. Although this method has been used to amplify protein and antibody-conjugated gold nanoparticles in histochemical microscopy studies (Hacker, in Colloidal Gold: Principles, Methods, and Applications, M.A. Hayat, Ed. (Academic Press, San Diego, 1989)), vol.1, chap.10; Zehbe et al., Am.J.Pathol.150, 1553 (1997)), but its use in quantitative DNA hybridization assays is new (Tomlinson et al., Anal.Biochem. , 171:217 (1988)). This method not only enables nanoparticle probes with very low surface coverage to be visualized by flatbed scanners or with the naked eye, but also allows quantitative hybridization of target hybridization based on silver-stained amplification of scattered light from gold probes on stained regions. Significantly, the signal intensity obtained from samples containing methicillin-resistant S. aureus genomic DNA was higher than that obtained from samples containing methicillin-sensitive S. Much higher. This shows that this detection methodology can be used for the specific detection of the mec A gene in the presence of complex bacterial genomic DNA background, see Figure 16. This result is an unusual feature of nanoparticle oligonucleotide conjugates that enables ultrasensitive and selective detection of nucleic acids. It should also be noted that this method does not require enzymatic targets or signal amplification methods, and it provides a new way to detect genes from bacterial genomic DNA samples.

(a) Target DNA preparation

Purified total genomic DNA isolated from Staphylococcus bacterial cells was purchased from ATCC (American Type Culture Collection). Prior to array hybridization, total genomic DNA was fragmented by sonication to shear DNA molecules as described in Example 5 (see below).

(b) MecA gene detection test

(ii) Test method

Reaction mixtures of bacterial genomic DNA and 1 nM nanoparticle probes in the range of 250 ng-1 μg were prepared in 1× hybridization buffer (5×SSC, 0.05% Tween 20). The reaction mixture was heated to 95°C for 5 minutes. Subsequently, 10-25 μL of the reaction mixture was added to the surface of the microarray, and hybridized at 40° C. and 90% relative humidity for 2 hours. The microarray surface was washed with 5×SSC, 0.05% Tween 20 for 30 seconds at room temperature. Microarrays were dried and subjected to silver contact development for 4 minutes with commercial grade silver enhancer solution (Silver Enhancer Kit, Catalog No. SE-100, Sigma, St. Louis). Silver-stained microarray plates were then washed, dried and imaged with an Arrayworx(R) scanner (Model No. AWE, Applied Precision, Inc., Issaquah, WA).

Example 5

Staphylococcal Speciation Analysis Using Bacterial Genomic DNA and Gold Nanoparticle-Labeled Tuf Probes

In this example, staphylococcal speciation assays were performed by identifying the Tuf gene sequences corresponding to S. aureus and S. epidermidis species. The Tuf 372bp PCR amplicon amplified from total genomic DNA isolated from S. aureus and S. epidermidis bacterial cells served as a positive control to demonstrate the sequence specificity of the array. In separate hybridization reactions, total genomic DNA isolated from S. aureus and S. epidermidis bacterial cells was fragmented and hybridized to microarray plates. The microarray plate includes Tuf 3, Tuf 4, or Tuf 5 and Tuf 6 capture probes bound thereto. Tuf 2 oligonucleotide-labeled gold nanoparticles were used as detection probes. Microarray plates, capture and detection probes were prepared as described in Example 3. The Tuf 372bp amplicon was prepared by traditional PCR amplification method.

(a) Target DNA preparation

Genomic DNA was prepared as follows: Genomic DNA isolated from cultured Staphylococcus bacterial cells was purchased from ATCC (American Type Culture Collection). The >10 μg portion of dried DNA was rehydrated in a volume of 200 μl of DNase-free water. It was then sonicated using a Misonix, Ultrasonic cell disruptor XL Farmingdale, NY, with 12 pulses of 2 watts of power for approximately 0.5 seconds each. Total DNA concentration was determined using a commercially available Picogreen kit from Molecular Probes and read on a Tecanspectrafluor plus fluorescent plate reader. Scanning analysis (smear analysis) was carried out on an Agilent 2100 bioanalyzer (Bioanalyzer), and the size of the DNA fragments thus measured was an average of 1.5Kb. A 372 base pair PCR amplicon of the positive control tuf gene was prepared from the genomic DNA of S. aureus or S. epidermidis using traditional PCR amplification techniques.

(b) Tuf gene detection test method

In separate hybridization wells, fragmented total genomic DNA isolated from Staphylococcus epidermidis or Staphylococcus aureus bacterial cells (8.0E07 copies, ~250ng) and 1nM nanoparticle probes were mixed in 1x hybridization buffer (5X SSC , 0.05% Tween 20). The PCR-amplified Tuf gene fragment of the same genomic DNA sample was used as a positive control, mixed with probe and buffer in a separate hybridization well on a glass slide. The reaction mixture was heated to 95°C for 5 minutes. Subsequently, 50 μl of the reaction mixture was added to the surface of the microarray and hybridized for 1.5 hours at 45° C. and 90% relative humidity. Microarray surfaces were rinsed in 0.5M _NaNO3 for 30 seconds at room temperature. The microarrays were dried and subjected to silver contact development for 4 minutes with a commercial grade silver enhancer solution (Silver Enhancer Kit, Catalog No. SE-100, Sigma, St., Louis, MO). The silver-stained microarray plate was then rinsed, dried, and scattered light from the silver-stained amplifying nanoparticle probes on the array was imaged and quantified using an Arrayworx(R) scanner (Model No. AWE, Applied Precision, Issaquah, WA).

The results for the Tuf3 and Tuf4 capture probes are shown in Figure 17(a) and (b), and the results for the Tuf5 and Tuf6 capture probes are shown in Figure 17(c) and (d). Using the Tuf3 and Tuf4 capture probe sets, specific signals on the array corresponding to the Staphylococcus species S. aureus and S. epidermidis were observed when genomic DNA was hybridized to the array. This strongly demonstrates that, in the presence of total genomic DNA, complexity reduction and amplification of tuf gene targets by PCR are not required to distinguish these closely related sequences. Using the Tuf3 and Tuf4 capture probe sets, signals corresponding to the appropriate species were also observed, but with some cross-reactivity with the mismatch capture sequences, resulting in lower recognition rates. This suggests that sequence design is critical for the precise identification of species.

Example 6

Staphylococcal speciation assay and methicillin resistance testing using PCR amplicons and gold nanoparticle-labeled mec A and Tuf oligonucleotides as detection probes

In this example, the array designed to identify the genus, species, and antibiotic resistance status of Staphylococcus was composed of 16S rRNA gene (genus), Tuf gene (Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus saprophyticus). The sequences of the species-specific capture species) and the mec A gene (antibiotic resistance status) were made. Note that the S. epidermidis and S. saprophytic capture probes differ by only one nucleotide, while the S. aureus and S. epidermidis capture probes differ by three nucleotides. The microarray plate includes all of the following sequences: 16S 12, mecA 6, Tuf 3, Tuf 4, Tuf 10 capture probes and one negative hybridization capture probe bound thereto. Gold nanoparticle-labeled Tuf 2, mecA 4, and 16S 12 probes were used as detection probes. Microarray plates, capture probes and detection probes were prepared according to the method described in Example 4. The specificity of the arrays was tested with PCR amplified gene sequences from various methicillin-resistant and methicillin-sensitive Staphylococcus species (S. aureus, S. epidermidis, S. saprophyticus). The specific PCR amplified gene fragments used for testing are shown in Figure 18 (mecA 281, 16S 451, and Tuf 372). Tuf gene sequences derived from the different Staphylococcus species shown in Figure 18 were obtained from GenBank.

Target preparation:

PCR amplified gene products were prepared using standard PCR amplification methods.

test:

Each reaction included 50 μl of 5x SSC, 0.05% Tween 20, 0.01% BSA, 200 pM of each nanoparticle probe, and 15% formamide and 750 pM of each target amplicon. The reagents were hybridized for 1 hour at 40°C and 90% humidity. Wash the microarray surface in 0.5 M NaNO ₃ for 30 s at room temperature. The microarrays were dried and subjected to silver contact development for 4 minutes with a commercial grade silver enhancer solution (Silver Enhancer Kit, Catalog No. SE-100, Sigma, St. Louis, MO). Silver-stained microarray plates were then rinsed, dried, and imaged with an Arrayworx(R) scanner (Model No. AWE, Applied Precision, Issaquah, WA).

The results are shown in Figure 19(a) and (b). The species and methicillin resistance status of the five selected Staphylococcus samples (see Table 3 below) were correctly identified with PCR amplicons showing array sequence specificity when standard PCR amplification methods were employed.

table 3 ATCC Sample ID# describe 35556700699122283598415305 Staphylococcus aureus, Mu50 - Methicillin-resistant Staphylococcus epidermidis, RP62A - Multi-antibiotic-resistant Staphylococcus saprophyticus

Example 7

Staphylococcal Speciation Analysis and Methicillin Resistance Testing Using Genomic DNA and Gold Nanoparticle-Labeled Probes for mecA, 16S, and Tuf

In this example, the identification of the genus, species and antibiotic resistance status of Staphylococcus was performed using total genomic DNA isolated from bacterial cells of S. aureus and S. epidermidis. The tested genomic DNA samples were characterized by ATCC as described in Table 3 above. The microarray plate and detection probes used in the test in Example 6 were also used in this example. Microarray plates and capture and detection probes were prepared as described in Example 3. Genomic DNA samples were prepared according to the method in Example 5. Each reaction included 50 μl of 5x SSC, 0.05% Tween 20, 0.01% BSA, 200 pM of each nanoparticle probe, and 15% formamide and 3.3 ng/μl of sonicated genomic DNA. The reagents were hybridized for 2 hours at 40°C and 90% humidity. Wash the microarray surface in 0.5 M NaNO ₃ for 30 s at room temperature. The microarrays were dried and subjected to silver contact development for 4 minutes with a commercial grade silver enhancer solution (Silver Enhancer Kit, Catalog No. SE-100, Sigma, St., Louis). Silver-stained microarray plates were then rinsed, dried, and imaged using an Arrayworxt(R) scanner (Model No. AWE, Applied Precision, Issaquah, WA).

The result is shown in Figure 20. Clearly, the Staphylococcus species and antibiotics were correctly identified for the three genomic DNAs tested based on net signal intensities in each sample that were only three standard deviations above background at the correct capture probe sites resistance status. This experiment showed that even single nucleotide mutations within the Tuf gene could be detected when staphylococcal genomic DNA was hybridized to the array and labeled with silver-stained amplified gold nanoparticle probes. Thus, by this new detection methodology that does not require any enzyme-based target amplification (such as PCR) or signal amplification (horseradish peroxidase) methods, it is possible to identify differences as small as a single nucleotide in a given gene sequence. Biological microorganisms for speciation analysis.

A known amount of total genomic DNA isolated from methicillin-resistant Staphylococcus aureus cells was added dropwise to the test system (assay), and the net signal intensity from the mecA gene capture probe was measured, as shown in Figure 21, to measure Test sensitivity. The lowest detectable amount was 34 ng, corresponding to approximately 10 million genome copies. Further optimization of the described detection method should enable the detection of lower amounts of genomic DNA.

It should be understood that the foregoing disclosure focuses on certain specific embodiments of the invention, and that all modifications or substitutions equivalent thereto are included within the spirit and scope of the invention, as set forth in the appended claims.

Claims (167)

1. A method for detecting a target nucleic acid sequence in a sample comprising nucleic acid molecules of higher biological complexity relative to amplified nucleic acid molecules, the target nucleic acid sequence differing from one or more nucleic acid sequences by at least one nucleotide, the The method includes the steps of: a) providing an addressable substrate bound to a capture oligonucleotide, wherein the capture oligonucleotide has a sequence complementary to at least a portion of the first portion of the target nucleic acid sequence; b) providing a detection probe comprising a detection oligonucleotide, wherein the detection oligonucleotide has a sequence complementary to at least a portion of the second portion of the target nucleic acid sequence of step (a); c) where the capture oligonucleotide is capable of effectively hybridizing to a first portion of the target nucleic acid sequence, the detection probe is capable of effectively hybridizing to a second portion of the target nucleic acid sequence, and allows for the discrimination of the target sequence from said target sequence having at least one nucleotide difference contacting the sample with the substrate and detection probes under conditions of one or more nucleic acid sequences; and d) detecting whether the capture oligonucleotide and the detection probe hybridize to the first and second portions of the target nucleic acid sequence. 2. The method of claim 1, wherein the target nucleic acid sequence comprises a single nucleotide polymorphism. 3. The method of claim 1, wherein a single nucleotide difference is recognized by a capture oligonucleotide bound to the substrate. 4. The method of claim 1, wherein a single nucleotide difference is recognized by a detection oligonucleotide. 5. The method of claim 1, wherein the target nucleic acid molecule comprises genomic DNA, genomic RNA, expressed RNA, plasmid DNA, mitochondrial or other organelle DNA, free cellular DNA, viral DNA or viral RNA, or two or both of the above more than one mixture. 6. The method of claim 1, wherein the substrate comprises a plurality of capture oligonucleotides, each of which is capable of recognizing a different single nucleotide polymorphism. 7. The method of claim 1, wherein the sample comprises more than one nucleic acid target, wherein each nucleic acid target comprises one or more different single nucleotide polymorphisms. 8. The method of claim 1, wherein one or more types of detection probes are provided, each detection probe having associated therewith a detection oligonucleotide capable of hybridizing to a different nucleic acid target. 9. The method of claim 1, wherein the sample is contacted with the detection probe so that the nucleic acid target present in the sample hybridizes with the detection oligonucleotide on the detection probe, and then the nucleic acid target bound to the detection probe is hybridized. The object is contacted with the substrate to hybridize the nucleic acid target to the capture oligonucleotide on the substrate. 10. The method of claim 1, wherein the sample is contacted with the substrate to hybridize the nucleic acid target present in the sample with the capture oligonucleotide, and then the nucleic acid target bound to the capture oligonucleotide is contacted with the detection probe to hybridize the nucleic acid target to the detection oligonucleotide on the detection probe. 11. The method of claim 1, wherein the sample is contacted with the probe probe and the substrate simultaneously. 12. The method of claim 1, wherein the detection oligonucleotide comprises a detectable label. 13. The method of claim 12, wherein the detection label allows detection by photonic, electronic, acoustic, photoacoustic, gravitational, electrochemical, electro-optic, mass spectrometric, enzymatic, chemical, biochemical, or physical means probing. 14. The method of claim 12, wherein the label is fluorescent. 15. The method of claim 12, wherein the label is luminescent. 16. The method of claim 12, wherein the label is phosphorescent. 17. The method of claim 12, wherein the label is radioactive. 18. The method of claim 12, wherein the label is a nanoparticle. 19. The method of claim 12, wherein the label is a dendrimer. 20. The method of claim 12, wherein the marker is a molecular aggregate. 21. The method of claim 12, wherein the label is a quantum dot. 22. The method of claim 12, wherein the markers are beads. 23. The method of claim 1, wherein the detection probe is a nanoparticle probe bound to a detection oligonucleotide. 24. The method of claim 23, wherein the nanoparticles are made of a noble metal. 25. The method of claim 24, wherein the nanoparticles are made of gold or silver. 26. The method of claim 25, wherein the nanoparticles are made of gold. 27. The method of claim 23, wherein detecting comprises contacting the substrate with a silver stained area. 28. The method of claim 23, wherein detecting comprises detecting light scattered by the nanoparticles. 29. The method of claim 23, wherein detecting comprises observing with an optical scanner. 30. The method of claim 23, wherein detecting comprises viewing with a flatbed scanner. 31. The method according to claim 29 or 30, wherein the scanner is connected to a computer loaded with software capable of calculating a grayscale value which provides a quantitative value for the amount of detected nucleic acid. 32. The method of claim 23, wherein the oligonucleotide attached to the substrate is located between two electrodes, the nanoparticles are made of an electrically conductive material, and step (d) includes detecting a change in conductivity. 33. The method of claim 32, wherein the electrodes are made of gold and the nanoparticles are also made of gold. 34. The method of claim 32, wherein the substrate is brought into contact with the silver-stained region, resulting in a change in conductivity. 35. The method of claim 23, wherein each of a plurality of oligonucleotides capable of recognizing different target nucleic acid sequences is attached to an array of spots on the substrate, each oligonucleotide spot being positioned between two electrodes, nanometer The particles are made of an electrically conductive material, and step (d) includes detecting a change in electrical conductivity. 36. The method of claim 35, wherein the electrodes are made of gold and the nanoparticles are also made of gold. 37. The method of claim 35, wherein the substrate is contacted with the silver-stained region to produce a change in conductivity. 38. A method of detecting one or more target nucleic acid sequences in a sample comprising nucleic acid molecules of greater biological complexity than amplified nucleic acid molecules, each of the one or more target nucleic acid sequences being compatible with known nucleic acid sequences differ by at least one nucleotide, the method comprising the steps of: a) providing an addressable substrate bound to a plurality of capture oligonucleotides, wherein the capture oligonucleotides have a sequence complementary to one or more portions of one or more target nucleic acid sequences; b) providing one or more detection probes comprising a detection oligonucleotide, wherein the detection oligonucleotide has a sequence complementary to one or more portions of the one or more target nucleic acid sequences of step (a); c) where the capture oligonucleotides are capable of efficiently hybridizing to one or more portions of one or more target nucleic acid sequences, the detection probes are capable of efficiently hybridizing to portions of one or more target nucleic acid sequences, and allow discrimination of contacting the sample with the substrate and the detection probe under conditions in which the target nucleic acid sequence differs in nucleotides; and d) detecting whether any capture oligonucleotides and detection probes hybridize to any target nucleic acid sequences. 39. The method of claim 38, wherein the target nucleic acid sequence comprises a single nucleotide polymorphism. 40. The method of claim 38, wherein single nucleotide differences are recognized by capture oligonucleotides bound to the substrate. 41. The method of claim 38, wherein single nucleotide differences are identified by detection oligonucleotides. 42. The method of claim 38, wherein the target nucleic acid molecule comprises genomic DNA, genomic RNA, expressed RNA, plasmid DNA, mitochondrial or other organelle DNA, free cellular DNA, viral DNA or viral RNA, or two or both of the above more than one mixture. 43. The method of claim 38, wherein the substrate comprises a plurality of capture oligonucleotides, wherein each capture oligonucleotide is capable of recognizing a different single nucleotide polymorphism. 44. The method of claim 38, wherein the sample comprises more than one nucleic acid target, each of which comprises a different single nucleotide polymorphism. 45. The method of claim 38, wherein one or more types of detection probes are provided, wherein each nucleic acid target has associated detection oligonucleotides capable of hybridizing to a different nucleic acid target. 46. The method of claim 38, wherein the sample is contacted with the detection probe so that the nucleic acid target present in the sample hybridizes with the detection oligonucleotide on the detection probe, and then the nucleic acid target bound to the detection probe is hybridized. The object is contacted with the substrate to hybridize the nucleic acid target to the capture oligonucleotide on the substrate. 47. The method of claim 38, wherein the sample is contacted with the substrate to hybridize the nucleic acid target present in the sample with the capture oligonucleotide, and then the nucleic acid target bound to the capture oligonucleotide is contacted with the detection probe to hybridize the nucleic acid target to the detection oligonucleotide on the detection probe. 48. The method of claim 38, wherein the sample is contacted with the detection probe and the substrate simultaneously. 49. The method of claim 38, wherein the detection probe comprises a detectable label. 50. The method of claim 49, wherein the detection label permits detection by photonic, electronic, acoustic, photoacoustic, gravitational, electrochemical, electro-optical, mass spectrometric, enzymatic, chemical, biochemical, or physical means. 51. The method of claim 49, wherein the label is fluorescent. 52. The method of claim 49, wherein the label is luminescent. 53. The method of claim 49, wherein the label is phosphorescent. 54. The method of claim 49, wherein the label is radioactive. 55. The method of claim 49, wherein the label is a nanoparticle. 56. The method of claim 49, wherein the label is a dendrimer. 57. The method of claim 49, wherein the marker is a molecular aggregate. 58. The method of claim 49, wherein the label is a quantum dot. 59. The method of claim 49, wherein the markers are beads. 60. The method of claim 38, wherein the detection probe is a nanoparticle probe bound to a detection oligonucleotide. 61. The method of claim 60, wherein the nanoparticles are made of a noble metal. 62. The method of claim 61, wherein the nanoparticles are made of gold or silver. 63. The method of claim 62, wherein the nanoparticles are made of gold. 64. The method of claim 60, wherein detecting comprises contacting the substrate with a silver stained area. 65. The method of claim 60, wherein detecting comprises observing light scattered by the nanoparticles. 66. The method of claim 60, wherein detecting comprises observing with an optical scanner. 67. The method of claim 60, wherein detecting comprises viewing with a flatbed scanner. 68. The method of claim 66 or 67, wherein the scanner is connected to a computer loaded with software capable of calculating a grayscale value that provides a quantitative value for the amount of nucleic acid detected. 69. The method of claim 60, wherein the oligonucleotide attached to the substrate is located between two electrodes, the nanoparticles are made of an electrically conductive material, and step (d) includes detecting a change in conductivity. 70. The method of claim 69, wherein the electrodes are made of gold and the nanoparticles are also made of gold. 71. The method of claim 69, wherein the substrate is contacted with the silver-stained region to produce a change in conductivity. 72. The method of claim 60, wherein each of a plurality of oligonucleotides capable of recognizing different target nucleic acid sequences is attached to an array of spots on the substrate, each oligonucleotide spot being positioned between two electrodes, nanometer The particles are made of an electrically conductive material, and step (d) includes detecting a change in electrical conductivity. 73. The method of claim 72, wherein the electrodes are made of gold and the nanoparticles are also made of gold. 74. The method of claim 72, wherein the substrate is contacted with the silver-stained region to produce a change in conductivity. 75. A method of identifying single nucleotide polymorphisms in a sample comprising nucleic acid molecules of greater biological complexity than amplified nucleic acid molecules, the method comprising the steps of: a) providing an addressable substrate bound to at least one capture oligonucleotide, wherein said at least one capture oligonucleotide has a sequence complementary to at least a portion of a nucleic acid target comprising a particular polymorphism; b) providing a detection probe bound to a detection oligonucleotide, wherein the detection oligonucleotide has a sequence complementary to at least a portion of the nucleic acid target of step (a); c) subjecting the sample to the substrate and the detection probe under conditions that allow efficient hybridization of the capture oligonucleotide to the nucleic acid target, efficient hybridization of the detection probe to the nucleic acid target, and allow discrimination of targets having a single nucleotide difference. pin contact; and d) Detecting whether the capture oligonucleotide and detection probe hybridize to the nucleic acid target. 76. The method of claim 75, wherein the polymorphism is recognized by a capture oligonucleotide bound to the substrate. 77. The method of claim 75, wherein the polymorphism is identified by a probe oligonucleotide. 78. The method of claim 75, wherein the nucleic acid molecules in the sample comprise genomic DNA, genomic RNA, expressed RNA, plasmid DNA, mitochondrial or other organelle DNA, free cellular DNA, viral DNA or viral RNA, or both or a mixture of two or more. 79. The method of claim 75, wherein the substrate comprises a plurality of capture oligonucleotides, wherein each capture oligonucleotide is capable of recognizing a different single nucleotide polymorphism. 80. The method of claim 75, wherein the sample comprises more than one nucleic acid target, wherein each nucleic acid target comprises one or more different single nucleotide polymorphisms. 81. The method of claim 75, wherein one or more types of detection probes are provided, wherein each detection probe incorporates a detection oligonucleotide capable of hybridizing to a different nucleic acid target. 82. The method of claim 75, wherein the sample is contacted with the detection probe so that the nucleic acid target present in the sample hybridizes to the detection oligonucleotide on the detection probe, and then the nucleic acid target bound to the detection probe is hybridized. The object is contacted with the substrate to hybridize the nucleic acid target to the capture oligonucleotide on the substrate. 83. The method of claim 75, wherein the sample is contacted with the substrate to hybridize the nucleic acid target present in the sample with the capture oligonucleotide, and then the nucleic acid target bound to the capture oligonucleotide is contacted with the detection probe to hybridize the nucleic acid target to the detection oligonucleotide on the detection probe. 84. The method of claim 75, wherein the sample is contacted with the probe probe and the substrate simultaneously. 85. The method of claim 75, wherein the detection probe comprises a detectable label. 86. The method of claim 85, wherein the detection label permits detection by photonic, electronic, acoustic, photoacoustic, gravitational, electrochemical, electro-optic, mass spectrometric, enzymatic, chemical, biochemical, or physical means. 87. The method of claim 85, wherein the label is fluorescent. 88. The method of claim 85, wherein the label is a nanoparticle. 89. The method of claim 85, wherein the label is luminescent. 90. The method of claim 85, wherein the label is phosphorescent. 91. The method of claim 85, wherein the label is radioactive. 92. The method of claim 85, wherein the label is a dendrimer. 93. The method of claim 85, wherein the marker is a molecular aggregate. 94. The method of claim 85, wherein the label is a quantum dot. 95. The method of claim 85, wherein the markers are beads. 96. The method of claim 75, wherein the detection probe is a nanoparticle probe bound to a detection oligonucleotide. 97. The method of claim 96, wherein the nanoparticles are made of a noble metal. 98. The method of claim 97, wherein the nanoparticles are made of gold or silver. 99. The method of claim 98, wherein the nanoparticles are made of gold. 100. The method of claim 96, wherein detecting comprises contacting the substrate with a silver stained area. 101. The method of claim 96, wherein detecting comprises detecting light scattered by the nanoparticles. 102. The method of claim 96, wherein detecting comprises observing with an optical scanner. 103. The method of claim 96, wherein detecting comprises viewing with a flatbed scanner. 104. The method of claim 102 or 103, wherein the scanner is connected to a computer loaded with software capable of calculating a grayscale value that provides a quantitative value for the amount of detected nucleic acid. 105. The method of claim 96, wherein the oligonucleotide attached to the substrate is located between two electrodes, the nanoparticles are made of an electrically conductive material, and step (d) includes detecting a change in conductivity. 106. The method of claim 105, wherein the electrodes are made of gold and the nanoparticles are also made of gold. 107. The method of claim 105, wherein the substrate is contacted with the silver-stained region to produce a change in conductivity. 108. The method of claim 96, wherein a plurality of oligonucleotides each capable of recognizing a different single nucleotide polymorphism are attached to the substrate in an array of spots, each oligonucleotide spot being located on two electrodes Between, the nanoparticles are made of an electrically conductive material, and step (d) includes detecting a change in electrical conductivity. 109. The method of claim 108, wherein the electrodes are made of gold and the nanoparticles are also made of gold. 110. The method of claim 109, wherein the substrate is contacted with the silver-stained region to produce a change in conductivity. 111. A method of identifying one or more single nucleotide polymorphisms in a sample comprising nucleic acid molecules of greater biological complexity than amplified nucleic acid molecules, the method comprising the steps of: a) providing an addressable substrate bound to a plurality of capture oligonucleotides, wherein the capture oligonucleotides have sequences complementary to portions of a nucleic acid target, each of said portions comprising a particular polymorphism; b) providing one or more detection probes comprising a detection oligonucleotide, wherein the detection oligonucleotide has a sequence complementary to at least a portion of one of the nucleic acid targets of step (a) that is not detected by Recognized by capture oligonucleotides on the substrate; c) subjecting the sample and substrate and probing probe contact; and d) detecting whether any capture oligonucleotides and detection probes hybridize to any nucleic acid targets. 112. The method of claim 111, wherein the polymorphism is recognized by a capture oligonucleotide bound to the substrate. 113. The method of claim 111, wherein the polymorphism is identified by a probe oligonucleotide. 114. The method of claim 111, wherein the nucleic acid molecules in the sample comprise genomic DNA, genomic RNA, expressed RNA, plasmid DNA, mitochondrial or other cellular organelle DNA, free cellular DNA, viral DNA or viral RNA, or both or a mixture of two or more. 115. The method of claim 111, wherein the substrate comprises a plurality of capture oligonucleotides, each of which is capable of recognizing one or more different single nucleotide polymorphisms. 116. The method of claim 111, wherein the sample comprises more than one nucleic acid target, each of which comprises a different single nucleotide polymorphism. 117. The method of claim 111, wherein one or more types of detection probes are provided, each of which is bound to a detection oligonucleotide capable of hybridizing to a different nucleic acid target. 118. The method of claim 111, wherein the sample is contacted with the detection probe so that the nucleic acid target present in the sample hybridizes to the detection oligonucleotide on the detection probe, and then the nucleic acid target bound to the detection probe is hybridized. The object is contacted with the substrate to hybridize the nucleic acid target to the capture oligonucleotide on the substrate. 119. The method of claim 111 , wherein the sample is contacted with a substrate to hybridize the nucleic acid target present in the sample to the capture oligonucleotide, and then the nucleic acid target bound to the capture oligonucleotide is contacted with the detection probe to hybridize the nucleic acid target to the detection oligonucleotide on the detection probe. 120. The method of claim 111, wherein the sample is contacted with the probe probe and the substrate simultaneously. 121. The method of claim 111, wherein the detection oligonucleotide comprises a detectable label. 122. The method of claim 121, wherein the detection label permits detection by photonic, electronic, acoustic, photoacoustic, gravitational, electrochemical, electro-optical, mass spectrometric, enzymatic, chemical, biochemical, or physical means. 123. The method of claim 121, wherein the label is fluorescent. 124. The method of claim 121, wherein the label is luminescent. 125. The method of claim 121, wherein the label is phosphorescent. 126. The method of claim 121, wherein the label is radioactive. 127. The method of claim 121, wherein the label is a nanoparticle. 128. The method of claim 121, wherein the label is a dendrimer. 129. The method of claim 121, wherein the marker is a molecular aggregate. 130. The method of claim 121, wherein the label is a quantum dot. 131. The method of claim 121, wherein the markers are beads. 132. The method of claim 111, wherein the detection probe is a nanoparticle probe bound to a detection oligonucleotide. 133. The method of claim 132, wherein the nanoparticles are made of a noble metal. 134. The method of claim 133, wherein the nanoparticles are made of gold or silver. 135. The method of claim 134, wherein the nanoparticles are made of gold. 136. The method of claim 132, wherein detecting comprises contacting the substrate with a silver stained area. 137. The method of claim 132, wherein detecting comprises detecting light scattered by the nanoparticles. 138. The method of claim 132, wherein detecting comprises observing with an optical scanner. 139. The method of claim 132, wherein detecting comprises viewing with a flatbed scanner. 140. The method of claim 138 or 139, wherein the scanner is connected to a computer loaded with software capable of calculating a grayscale value that provides a quantitative value for the amount of nucleic acid detected. 141. The method of claim 132, wherein the oligonucleotide attached to the substrate is located between two electrodes, the nanoparticles are made of an electrically conductive material, and step (d) includes detecting a change in conductivity. 142. The method of claim 141, wherein the electrodes are made of gold and the nanoparticles are also made of gold. 143. The method of claim 141, wherein the substrate is contacted with the silver-stained region to produce a change in conductivity. 144. The method of claim 141, wherein a plurality of oligonucleotides each capable of recognizing a different single nucleotide polymorphism are attached to an array of spots on the substrate, each oligonucleotide spot being located on two electrodes Between, the nanoparticles are made of an electrically conductive material, and step (d) includes detecting a change in electrical conductivity. 145. The method of claim 144, wherein the electrodes are made of gold and the nanoparticles are also made of gold. 146. The method of claim 146, wherein the substrate is contacted with the silver-stained region to produce a change in conductivity. 147. The method of claim 1, 38, 75, or 111, wherein the higher biological complexity is greater than about 50,000. 148. The method of claim 1, 38, 75, or 111, wherein the higher biological complexity is about 50,000-50,000,000,000. 149. The method of claim 1, 38, 75, or 111, wherein the higher biological complexity is about 1,000,000,000. 150. The method of claim 1 or 38, wherein the target nucleic acid sequence is part of a biological microorganism gene. 151. The method of claim 1 or 38, wherein the nucleic acid sequence of interest is part of a staphylococcal gene. 152. The method of claim 151, wherein the Staphylococcus is Staphylococcus aureus, Staphylococcus haemolyticus, Staphylococcus epidermidis, Staphylococcus leonis, Staphylococcus hominis, or Staphylococcus saprophyticus. 153. The method of claim 151, wherein the nucleic acid sequence of interest is a portion of the Tuf gene. 154. The method of claim 151, wherein the nucleic acid sequence of interest is part of the femA gene. 155. The method of claim 151, wherein the target nucleic acid sequence is a part of the 16S rRNA gene. 156. The method of claim 151, wherein the nucleic acid sequence of interest is part of the hsp60 gene. 157. The method of claim 151, wherein the nucleic acid sequence of interest is part of the sodA gene. 158. The method of claim 1 or 38, wherein the target nucleic acid sequence is part of the mecA gene. 159. The method of claim 1 or 38, wherein the target nucleic acid sequence comprises SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 , SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 , SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56 , SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, a sequence set forth in SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, or SEQ ID NO: 78. 160. The method of claim 1 or 38, wherein at least one of the probe oligonucleotides comprises SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38 , SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55 , SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, or a sequence set forth in SEQ ID NO: 78. 161. The method of claim 1, wherein the capture oligonucleotide comprises SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO : 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO : 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48 , SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO : 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, or a sequence listed in SEQ ID NO: 78. 162. The method of claim 38, wherein at least one of the capture oligonucleotides comprises SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, or a sequence listed in SEQ ID NO: 78. 163. The method of claim 38, wherein at least one of the target nucleic acid sequences is part of a staphylococcal gene and at least one of the target nucleic acid sequences is part of a mecA gene. 164. The method of claim 38, wherein the method is used to distinguish between two or more species within the same genus. 165. The method of claim 164, wherein the species differ by two or more discrete nucleotides. 166. The method of claim 164, wherein the species differ by two or more consecutive nucleotides. 167. The method of claim 164, wherein said species differ by at least one nucleotide.

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