CN1878874A - Direct SNP detection with unamplified DNA - Google Patents

Abstract

The present invention provides methods for detecting a target nucleic acid molecule in a sample that comprises nucleic acid molecules of higher biological complexity than that of amplified nucleic acid molecules. In particular, the present invention provides methods and probes for detecting a single nucleotide polymorphism (SNP) in a sample that comprises nucleic acid molecules of higher biological complexity than that of amplified nucleic acid molecules.

Description

Use the direct SNP of non-DNA amplification to detect

The U.S. provisional application No.60/432 that the application and on December 12nd, 2002 submit to, 772 and No.60/433,442 is relevant, and require its right of priority, and its disclosed content is in the lump at this as a reference.

Technical field

The present invention relates to the method for target nucleic acid molecules in the test sample, comprise nucleic acid molecule in this sample, for example in genomic dna with biological complex degree higher than the nucleic acid molecule of amplification.Particularly, the present invention relates to use method and the probe of the probe in detecting SNP of nanometer particle to mark.The present invention also relates to the detection of biological organism, be specially bacterial pathogens such as staphylococcus DNA and detect antibiotics resistant gene in the sample, as the method for giving the mecA gene of microbiotic X-1497 (methicillin) resistance.

Background technology

Single nucleotide polymorphism between the genomic dna that observes in Different Individual (SNPs) or single nucleotide variation have not only constituted the basis of genetic diversity, the mark that also is considered to the disease tendency can be used for carrying out disease control better, increases more efficient drug is found in the understanding and the final promotion of disease condition.Therefore, for this common target, i.e. development can be differentiated the method for SNPs fast and reliablely, is carrying out a large amount of effort.Because human genome DNA's inherent complexity (monoploid genome=3 * 10 ⁹Bp) with the needs of relevant susceptibility, most these effort all need by carrying out target amplification as methods such as PCR.The direct ability that detects SNPs in the human genome DNA will make to detect to be oversimplified, and eliminates the error relevant with target amplification in the SNP discriminating.

Can adopt many methods to identify single nucleotide polymorphism, comprise dna sequencing, Restriction Enzyme analysis or the hybridization of specificity site.But,, need highly accurately and delicately to analyze simultaneously the ability in a plurality of sites to the screening of the high-throughput genome range of SNP and sudden change.In order to improve sensitivity and specificity, the high throughput method that detects mononucleotide at present depends on the step that relates to the target nucleic acid sample amplification, common employing polymerase chain reaction (PCR) (referring to, for example, Nikiforov etc., be published in the U.S.Pat.No.5 on October 21st, 1997,679,524; McIntosh etc., the PCT that is published on December 30th, 1998 applies for WO 98/59066; Goelet etc., the PCT that is published in May 11 nineteen ninety-five applies for WO 95/12607; Wang etc., 1998, Science 280:1077-1082; Tyagi etc., 1998, Nature Biotechnol.16:49-53; Chen etc., 1998, Genome Res.8:549-556; Pastinen etc., 1996, Clin.Chem.42:1391-1397; Chen etc., 1997, Proc.Natl.Acad.Sci.94:10756-10761; Shuber etc., 1997, Hum.Mol.Gen.6:337-347; Liu etc., 1997, Genome Res.7:389-398; Livak etc., Nature Genet.9:341-342; Day and Humphries, 1994, Anal.Biochem.222:389-395).Why pcr amplification is necessary to the conventional hybridization based on SNP, mainly contains two reasons: the first, have 3 when obtaining each monoploid genome, 000, during the total DNA of the people of 000,000 base pair, the target sequence that contains the SNP site has only been represented the very little part of total DNA.For example, the target sequence of 20 base pairs has only been represented 0.00000033% (normal genome has two target sequence copies, but they have different SNP sites, and therefore is considered to different sites) of total DNA.Therefore, because the deficiency of sensitivity, the common DNA sample of minority microorganism is not enough for many prior aries.Yet prior reason is, the enough short so that oligonucleotide that allows to distinguish single base is not to hybridize with the target area with the hybridization of the target sequence of 20 bases single-mindedly, but combines with other zone in the genome on less degree.Because the overwhelming quantity of non-target dna, non-specific hybridization has been made very big background, so that covered specific signals.Therefore, the amplification of a target area has just become the steps necessary of the non-specific sequence of remarkable minimizing.This amplification step is called " reduced complexity (complexity reduction) ".Yet the fidelity of reproduction of round pcr is limited.The right combination of PCR primer is tended to produce the pseudoreaction product or is unsuccessful in some specific regions.In addition, after non-target sequence was replicated, when perhaps making mistake be imported into target sequence owing to erroneous combination, the wrong number in the end product just rose along with each round-robin pcr amplification is index.Like this, during rare variation in searching nucleic acid population, the PCR mistake just may be a basic shortcoming.

At last, using the shortcoming of target amplification is all separately amplifications of each SNP site.And owing to have millions of SNP in the human genome, therefore, this becomes can't finishing of task.Even if the method for amplification and strategy are around SNP-locus specificity amplification problem, current state-of-art also can only identify simultaneously very small proportion (being less than 0.1%) among total SNP (see, for example, Kennedy etc., 2003, Nature Biotechnol.21:1233-1237).The Eric Lander of Whitehead biomedical research institute (Whitehead Institute for Biomedical Reserch) and one of them leader of the Human Genome Project point out, give up target amplification and be one of challenge the most great in the genome range SNP screening (seeing Lander E.1999, Nature Genetics Suppl.21:3-4).Therefore, also need the method for SNP in the test sample sensitive more, effective, that cost is low in the prior art, such method does not need target amplification or reduced complexity.

The evaluation of dna mutation also is importantly (to see Edwards etc., J.Clin.Micro.39:3047-3051) for the evaluation of microorganism.For example, Staphylococcus comprises at least 38 different kinds, and the quilt evaluation in hospital infection of the great majority in these kinds (Edwards etc., J.Clin.Micro.39:3047-3051).Therefore, Rapid identification of microorganism and species form analyzes (speciation) to identifying that the source of infection is important, the evaluation of the source of infection helps to determine patient's methods of treatment and the intersection of the outbreak of identification infection on epidemiological significance and nosocomial pathogens is propagated (Olive and Bean, 1999, J.Clin.Micro.37:1661-1669).Long (1＞day) and usually can't accurately differentiate concrete species (Hamels etc., 2001, Biotechniques 31:1364-1372).Therefore, concrete bacterial species discrimination method, especially nosocomial pathogens such as the staphylococcus based on the nucleotide sequence evaluation that development is quick more, accurate and cost is littler all is absorbed in a large amount of work.The microorganism of identical family or genus comprises the gene (Hamels etc., 2001, Biotechniques 31:1364-1372) that conservative coding same protein takes place to go up in system.Although from the gene order of identical family high conservative normally, but identified specific specificity series jump among the several genes (as 16S rRNA).The PCR in real time that is used for the oligonucleotide probe that has developed target 16S rRNA gene variable region detect identify multiple coagulase-negative and positive staphylococcus species (Edwards etc., J.Clin.Micro.39:3047-3051).

In addition, developed microarray and identified Staphylococcus, kind and antibiotics resistance (Hamels etc., 2001, Biotechniques 31:1364-1372) by the femA gene order of pcr amplification.Microarray comprises the oligonucleotide probe of specific specificity sequence variations (sequence variations of three or more bases) in the identification femA gene, the femA gene is relevant with 5 kinds of clinical relevant staphylococcus bacterial classifications (streptococcus aureus (S.aureus), staphylococcus epidermidis (S.epidermidis), staphylococcus haemolyticus (S.haemolyticus), human-like staphylococcus (S.hominis), Staphylococcus saprophyticus (S.saprophyticus)), and the oligonucleotide probe of target homologous genes conserved regions is used for the discriminating of Staphylococcus.Yet, microarray and need PCR exactly based on a main shortcoming of the detection of PCR in real time, this makes it no matter is from clinical or from the viewpoint of cost not ideal (seeing that the PCR that the above-mentioned SNP of being used for identifies discusses).Therefore, still need in the prior art more sensitive, effectively, lower being used for of cost detects and the biology method of microorganism of analytic sample, such method does not need target amplification or reduced complexity.

Summary of the invention

The invention provides the method for target nucleic acid sequence in the test sample, wherein sample comprises the nucleic acid molecule than the higher biological complex degree of nucleic acid molecule of amplification, and the target nucleic acid sequence has the different of a mononucleotide at least with known nucleic acid sequence.For example, the difference of a mononucleotide just can be a single nucleotide polymorphism.

On the one hand, the method that does not comprise target nucleic acid sequence in the test sample of early stage target amplification or reduced complexity comprises the steps: a) to provide the addressable that is combined with capture oligo substrate, and wherein capture oligo has at least a portion complementary sequence with the first part of target nucleic acid sequence; B) provide and comprise the exploratory probe of surveying oligonucleotide, wherein survey oligonucleotide have with step (a) at least a portion complementary sequence of second section of target nucleic acid sequence; C) can effectively hybridize with the first part of target nucleic acid sequence at capture oligo, and exploratory probe can with the second section of target nucleic acid sequence effectively under the condition of hybridization, sample is contacted with exploratory probe with substrate; Whether and d) detect capture oligo and exploratory probe hybridizes with first and second parts of target nucleic acid sequence.

On the other hand, the method that does not comprise target nucleic acid sequence in the test sample of early stage target amplification or reduced complexity comprises the steps: a) to provide the addressable that is combined with many capture oligos substrate, and wherein capture oligo has one or more part complementary sequences with the target nucleic acid sequence; B) provide and comprise the exploratory probe of surveying oligonucleotide, wherein survey oligonucleotide have with step (a) in not by one or more part complementary sequences of the target nucleic acid sequence of the identification of the capture oligo on the substrate; C) can effectively hybridize with one or more parts of target nucleic acid sequence at capture oligo, and exploratory probe can with one or more parts of the target nucleic acid sequence of the oligonucleotide identification of not being hunted down effectively under the condition of hybridization, sample is contacted with exploratory probe with substrate; And d) detect capture oligo and exploratory probe whether with the target nucleic acid sequence hybridization.

The present invention also provides the method for identifying single nucleotide polymorphism in the sample, wherein comprises the nucleic acid molecule than the higher biological complex degree of nucleic acid molecule of amplification in the sample.

On the one hand, the method that does not comprise single nucleotide polymorphism in the evaluation sample of early stage target amplification or reduced complexity comprises step: the addressable that is combined with at least one capture oligo substrate a) is provided, and wherein said at least one capture oligo has and at least a portion complementary sequence that comprises the nucleic acid target material of specific polymorphism; B) provide and be combined with the exploratory probe of surveying oligonucleotide, wherein survey oligonucleotide and have at least a portion complementary sequence with the nucleic acid target material of step (a); C) can effectively hybridize with nucleic acid target material at capture oligo, and exploratory probe can with nucleic acid target material effectively under the condition of hybridization, sample is contacted with exploratory probe with substrate; Whether and d) detect capture oligo and exploratory probe hybridizes with nucleic acid target material.

On the other hand, the method that does not comprise single nucleotide polymorphism in the evaluation sample of early stage target amplification or reduced complexity comprises step: the addressable that is combined with a plurality of capture oligos substrate a) is provided, wherein capture oligo has a plurality of part complementary sequences with nucleic acid target material, and each part all comprises specific polymorphism; B) provide and contain the exploratory probe of surveying oligonucleotide, wherein survey oligonucleotide and have at least a portion complementary sequence with the nucleic acid target material of step (a), nucleic acid target material is not discerned by the capture oligo on the substrate; C) can effectively hybridize with a plurality of parts of nucleic acid target material at capture oligo, and exploratory probe can with nucleic acid target material effectively under the condition of hybridization, sample is contacted with exploratory probe with substrate; Whether and d) detect capture oligo and exploratory probe hybridizes with nucleic acid target material.

In one embodiment, the nucleotide difference of target nucleic acid or single nucleotide polymorphism can be incorporated into the capture oligo on the substrate or survey oligonucleotide and discern.

In another embodiment, the target nucleic acid molecules in the sample comprises RNA, plasmid DNA, plastosome or other cell organelles DNA, free cell DNA, viral DNA or viral RNA or above-mentioned two or more the mixture of genomic dna, geneome RNA, expression.

In one embodiment, employed substrate can comprise many capture oligos in the method for the present invention, each capture oligo can be discerned one or more different single nucleotide polymorphism or nucleotide differences, sample can comprise more than one nucleic acid target material, each nucleic acid target material all comprises different single nucleotide polymorphism or nucleosides difference, can with one of them hybridization in many capture oligos.In addition, method of the present invention can provide the exploratory probe of one or more types, and the exploratory probe of each type all is combined with the detection oligonucleotide, can hybridize with different nucleic acid target material.

In one embodiment, sample can contact with exploratory probe, so that be present in nucleic acid target material in the sample and the detection oligonucleotide hybridization on the exploratory probe, then, can be with nucleic acid target material and the substrate contacts that is incorporated on the exploratory probe, so that the hybridization of the capture oligo on nucleic acid target material and the substrate.Randomly, sample can with substrate contacts so that the hybridization of the nucleic acid target material in the sample and capture oligo, then, can contact with exploratory probe with capture oligo bonded nucleic acid target material, so that the detection oligonucleotide hybridization on nucleic acid target material and the exploratory probe.In another embodiment, sample can while and exploratory probe and substrate contacts.

In another embodiment, survey oligonucleotide and can comprise detectable mark.This mark can be, for example, fluorescence, luminous, phosphorescent property, radioactive or nanoparticle can be connected in dendrimer (dendrimer), molecule aggregates (molecular aggregate), quantum dot (quantum dot) or pearl with surveying oligonucleotide.Described mark can be surveyed by for example photonics, electronics, acoustics, optoacoustic, gravity, electrochemistry, electric light, mass spectrum, zymetology, chemistry, biological chemistry or physics means.

In one embodiment, exploratory probe can be to be combined with the nanoparticle probe of surveying oligonucleotide.Nanoparticle can be made by noble metal such as gold or silver.Nanoparticle can use for example optics or flat bed scanner detection.Scanner can connect on computers, and the software that can calculate gray-scale value has been installed on the computer, and the gray-scale value that is calculated provides the quantitative values of detected nucleic acid amount.By gold and silver or other the metal nanoparticle positions that can impel automatic metal development (autometallography), can dye mode and the nanoparticle bonded substrate that detects in high sensitivity with target nucleic acid molecules by silver.Randomly, can detect and nanoparticle bonded substrate by the light that detects the nanoparticle scattering.

In another embodiment, the oligonucleotide that is attached to substrate can be placed between two electrodes, nanoparticle can be made by electrical conductor material, and the step of method of the present invention (d) can comprise the variation that detects electroconductibility.In another embodiment, wherein each the many oligonucleotide that can both discern different target nucleic acid sequences is attached in the array of spots on the substrate, each oligonucleotide spot is all between two electrodes, nanoparticle can be made by electrical conductor material, and the step of method of the present invention (d) can comprise the variation that detects electroconductibility.Electrode can be made by for example gold, and nanoparticle also is made of gold.Alternatively, substrate can dye district (silver stain) with silver and contacts to produce the variation of electroconductibility.

In another embodiment, method of the present invention can be used for distinguishing two or more generic species.On the one hand, these species can have the difference of two or more discontinuous kernel thuja acids.On the other hand, these species can have the difference of two or more continuous nucleotides.

In one embodiment, the target nucleic acid sequence of the present invention part that can be aureus gene.Aspect of this embodiment scheme, staphylococcus can be for example streptococcus aureus (S.aureus), staphylococcus haemolyticus (S.haemolyticus), staphylococcus epidermidis (S.epidermidis), Lyons staphylococcus (S.lugdunensis), human-like staphylococcus (S.hominis), Staphylococcus saprophyticus (S.saprophyticus).Therefore, method of the present invention can be used for staphylococcic species formation analyses (for example: distinguish different types of staphylococcus).

In another embodiment, the target nucleic acid sequence of the present invention part that can be mec A gene.Therefore, method of the present invention can be used to identify X-1497 (methicillin) resistant strain.

In another one embodiment of the present invention, the target nucleic acid sequence, capture oligo, and/or the detection oligonucleotide can comprise SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQID 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 IDNO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQID 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 IDNO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQID 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 IDNO: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 the listed sequence of SEQ ID NO:78.

To certain preferred embodiments and the more detailed description of claim, the concrete preferred embodiment of the present invention will be more obvious by following.

Description of drawings:

Fig. 1 is the present invention's one step hybrid method synoptic diagram.

Fig. 2 is the present invention's two steps hybrid method synoptic diagram.

Fig. 3 schematically shows the hybridization complex of the exploratory probe of nanometer particle to mark, the wild-type that is incorporated into substrate or mutant capture probe and wildtype target thing.In order to detect SNP, test is carried out under suitable experiment condition, can keep the complex body (left side) of intact coupling and stop the formation (right side) of the complex body that contains mispairing under this experiment condition.

Fig. 4 shows human genome DNA's [(a) part] or the salmon sperm DNA [(b) part] with not amplification, and the SNP of the factor V gene that carries out on the Superaldehyde  sheet of factor V gene trap probe with wild or sudden change (1691 G-〉A) detects.(c) part is in the presence of wild-type or mutant capture probe, carries out the summary figure of the detection signal strength analysis of human genome DNA and nonspecific salmon sperm DNA.

Fig. 5 is shown as and makes method of the present invention can distinguish two target nucleic acids of a Nucleotide (SNP site) difference and regulate the importance of hybridization conditions.

Fig. 6 (a) and (b) and (c) show can array of designs (sequence capture probe) and hybridization conditions, so that can under with an array and same hybridization conditions, detect more than one SNP type, under the condition that is not relying on input DNA, SNP between wild-type and the mutant DNA is distinguished becomes possibility.

Fig. 7 is presented on the CodeLink  slide glass, under different methane amide concentration conditions, with the SNP detection that non-amplification human gene group DNA ((a) part) carries out factor V mutator gene (1691 G-〉A), wild-type and mutant factor V gene trap probe have been arranged with the method for hybridization on the CodeLink  slide glass.(b) the part chart to human genome DNA's detection signal strength analysis summarize, this detection signal strength analysis is in the presence of wild-type or mutant capture probe, carries out after the hybridization under different methane amide concentration.

Fig. 8 is presented under the condition that the best is adjusted, and people's wild-type DNA only produces signal on wild-type probe, and people's mutant DNA only produces signal on the mutant capture probe.

Fig. 9 (a)-(d) shows the quantitative data of desirable (intermediary) hybridization conditions among Fig. 8.

Figure 10 (a) and (b) show and to use very small amount of (being less than 1 milligram) total DNA of people to distinguish SNP.Figure 10 has shown that also the design of capture oligo and the suitable coupling under the stringent condition are for catching (and detection) length of probe and importance of Nucleotide composition.

Figure 11 (a) and (b) show to use method of the present invention, carries out the result that SNP detects by on a slide glass 10 separately in the hybridization in genomic dna.Coupling and mispairing net signal strength criterion deviation do not have overlappingly in 10 hybridization, mean for each hybridization, can both determine to import the SNP genotype of DNA reliably.

Figure 12 (a) and (b) show to use method of the present invention to carry out the result that multiple SNP identifies in whole genome DNA wherein detects the genotype of factor V, factor II and mthfr gene.

Figure 13 (a) and (b) be presented at and carry out the result that multiple SNP identifies among the whole genome DNA of patient's sample GM16028, it has shown that method of the present invention identifies the genotypic ability of heterozygosis SNP of factor V, factor II and mthfr gene in the single individuality.

Figure 14 (a) and (c) be presented at and carry out the result that multiple SNP detects among the whole genome DNA of patient's sample GM00037, its show method of the present invention identify single individuality to a gene be wild (being factor V in this example), to another gene (being factor II in this example) be heterozygosis, be the ability of sudden change to the 3rd gene (being MTHFR in this example).

Figure 15 (a)-(f) shows that three different investigators implement the result of method of the present invention to two independent patient's samples.

Figure 16 (a)-(b) shows that use is fixed on mecA 2 and mecA 6 capture oligos on the sheet glass, specific detection result to the mecA gene that comes from aureus gene group DNA, wherein aureus gene group DNA is located away from X-1497 resistance (mecA+) aureus cell, and the golden nanometer particle of mecA 4 marks is as exploratory probe.The aureus gene group DNA that is located away from methicillin-sensitivity (mecA-) aureus cell is as negative control.With the mecA gene (being labeled as 281 base pair fragments (281 base-pairfragment labeled MRSA 281bp) of MRSA 281bp) of the pcr amplification of known quantity as positive control.(a) part shows the X-1497 resistant gene group DNA target thing (75-300 1,000,000 copies) that a series of scan images from the microarray hole, microarray hole contain quantity and do not wait, and positive control is identical with it with the negative control sample.(b) part is the chart of show sample data analysis.Draw the net signal of X-1497 resistance staphylococcus aureus gene group DNA by the signal that deducts corresponding negative control spot.In all lines, horizontal black line representative is with respect to three standard deviations of the negative control spot that contains methicillin-sensitivity staphylococcus aureus gene group DNA.This figure shows the specific detection from the mec A of total bacterial genomes DNA.

Figure 17 forms with pcr amplification (amplicon) that comes from streptococcus aureus and staphylococcus epidermidis (the ATCC numbering is respectively 700699 and 35984) or genomic dna illustration staphylococcus species and analyzes.In order to detect total genomic dna, before hybridization array, adopt the ultrasonic degradation method to make the DNA sample fragmentization.(a) part is a series of scan images from the microarray hole, and microarray contains Tuf 372bp amplicon or genomic dna (300ng ,～8.0E7 copy).Water (no target thing) is with comparing.Array board comprises combination Tuf 3 and Tuf 4 capture probes thereon.Tuf 2 probes of golden nanometer particle mark are as exploratory probe.(b) part provides the chart that the sample data shown in representative (a) part is analyzed.Horizontal black line representative is with respect to three standard deviations of background.(c) part Tuf 372bp amplicon or genomic dna (8.0E7 copy).Array board comprises combination Tuf 5 and Tuf 6 capture probes thereon.(d) part provides the chart that the sample data shown in representative (c) part is analyzed.Horizontal black line representative is with respect to three standard deviations of background.

Figure 18 (a)-(c) provides the sequence of pcr amplification of the staphylococcus epidermidis Tuf of the streptococcus aureus Tuf of streptococcus aureus mecA, the streptococcus aureus coa of 450 base pairs of 281 base pairs used among the embodiment 4-6, the streptococcus aureus Tuf of 142 base pairs, 372 base pairs and 372 base pairs.

Figure 19 (a)-(j) illustration is used the target thing of pcr amplification to carry out the staphylococcus species and is formed analysis and mecA gene test, and the target thing of pcr amplification is taken from commercially available aureus strain ATCC 35556, ATCC 35984, ATCC 12228, ATCC 700699 and ATCC 15305.(a), (c), (e), (g) and (i) part be a series of scan images from the microarray hole, microarray contains any one PCR product in the 16S, the Tuf that represent 5 genome samples or the mecA gene.(b), (d), (f) and (h) part be represent a series of charts of 5 sample datas analyses.In all lines, horizontal black line representative is with respect to three standard deviations of background.

Figure 20 (a)-(f) illustration is used the genomic dna target thing of ultrasonic degradation to carry out the staphylococcus species and is formed analysis and mec A detection, and genomic dna target thing is ATCC 35984, ATCC 700699 and ATCC 12228 from commercially available aureus strain.(a), (c) and (e) part be a series of scan images in microarray hole, microarray contains from any genomic dna among ATCC 35984, ATCC 700699 or the ATCC12228.Array board comprises 16S 12, mecA 6, Tuf 3, Tuf 4, Tuf 10 capture probes that are combined with negative hybridization contrast.16S 13, the mecA4 of golden nanometer particle mark and Tuf2 probe are as exploratory probe.(b), (d) and (f) part be represent a series of charts of three sample datas analyses.In all lines, horizontal black line representative is with respect to three standard deviations of background.

Figure 21 is for showing the chart that carries out the susceptibility boundary of mec A gene test with genomic dna target thing.Use the sequence in the table 3, under 5x SCC, 0.05%Tween 20,0.01%BSA, 15%v/v methane amide and 200pM nanoparticle probe condition, 45 ℃ 1.5 hours, with the data analysis of the mec A gene test of carrying out ATCC700699 genome sample.Figure 21 shows the detection boundary of (34ng total genomic dna) 330fM in the 50 μ l reaction systems.The representative of the sea line at 80 places is with respect to three standard deviations of background among the figure.

Implement the preferred embodiments of the invention:

Unless context has requirement in addition, singular references should comprise plural term, and plural term should comprise singular references.

Unless otherwise noted, following term should be understood to have following implication with the employed unanimity of this specification sheets:

" nucleotide sequence " used herein, " nucleic acid molecule ", perhaps " nucleic acid " refers to one or more oligonucleotide or the polynucleotide that define here." target nucleic acid molecules " used herein or " target nucleic acid sequence " is meant that oligonucleotide or polynucleotide, this oligonucleotide or polynucleotide comprise the sequence in the sample that the inventive method user will detect.

The meaning of term " polynucleotide " is the strand or the double-strandednucleic acid polymer of at least 10 base length herein.In certain embodiments, the Nucleotide that comprises polynucleotide can be ribonucleotide or deoxyribonucleotide or any modified forms among them.Described modification comprises base modification such as broxuridine, ribose is modified as Arabinoside and 2 ', 3 '-dideoxy ribose, and key is modified as thiophosphatephosphorothioate (phosphorothioate), phosphorodithioate (phosphorodithioate), seleno phosphoric acid ester (phosphoroselenoate), two seleno phosphoric acid ester (phosphorodiselenoate), phosphoroanilothioate, phoshoraniladate and phosphoramidate (phosphoroamidate) between nucleosides.Term " polynucleotide " especially comprises the DNA of strand and double chain form.

Term " oligonucleotide " refers to comprise the Nucleotide of natural generation herein, and the modified nucleotide that links together of the oligonucleotide key that produces by natural and/or non-natural.Oligonucleotide is the subclass of polynucleotide, comprises to be generally strand, to have 200 or the member of base length still less.In certain embodiments, long 10 to 60 bases of oligonucleotide.In certain embodiments, long 12,13,14,15,16,17,18,19 or 20 to 40 bases of oligonucleotide.Oligonucleotide can be strand or two strands, for example, and when being used for the structure of gene mutation body.About the sequence of coded protein, oligonucleotide of the present invention can be for having justice or antisense oligonucleotide.

Term " Nucleotide of natural generation " comprises deoxyribonucleotide and ribonucleotide.Term " Nucleotide of modification " comprises Nucleotide or the similar Nucleotide that glycosyl group is modified or replaces.Term " oligonucleotide key " comprises the oligonucleotide key of thiophosphatephosphorothioate, phosphorodithioate, seleno phosphoric acid ester, two seleno phosphoric acid ester, phosphoroanilothioate, phoshoraniladate, phosphoramidate and so on.Referring to, for example, LaPlanche etc., 1986, Nucl.Acids Res., 14:9081; Stec etc., 1984, J.Am.Chem.Soc., 106:6077; Stein etc., 1988, Nucl.Acids Res., 16:3209; Zon etc., 1991, Anti-Cancer Drug Design, 6:539; Zon etc., 1991, OLIGONUCLEOTIDES AND ANALOGUES:A PRACTICAL APPROACH, pp.87-108 (F.Eckstein, Ed.), Oxford University Press, Oxford England; Stec etc., U.S.Pat.No.5,151,510; Uhlmann and Peyman, 1990, Chemical Reviews, 90:543, wherein disclosed content at this in the lump as the reference of any purpose.Oligonucleotide can comprise detectable mark, so that can survey oligonucleotide or its hybridization.

Employed in the method for the present invention " addressable substrate " can be any surface that can oligonucleotide binding.Such surface includes but not limited to glass, metal, plastics or bag by the material of functional group, and wherein functional group is designed for oligonucleotide binding.Bag can be compared monolayers thick; In fact, bag can be comprised enough thick porous seepage material to form the three-dimensional structure of porous infiltration, and oligonucleotide can spread so far three-dimensional structure and surface incorporated therein.

Term used herein " capture oligo " refers to be incorporated into the oligonucleotide of substrate, it comprises can be with complementary nucleotide sequence or the assignment of genes gene mapping (promptly, in sample, hybridize) nucleotide sequence on target nucleic acid molecules, and target nucleic acid molecules is attached on the substrate by capture oligo in the mode of hybridization.Suitable but example nonrestrictive capture oligo comprises DNA, RNA, PNA, LNA or their combination.Capture oligo can comprise native sequences or composition sequence, has or do not have the Nucleotide of modification.

" exploratory probe " of the present invention can be to adhere to any carrier that one or more survey oligonucleotide, one of them or survey oligonucleotide more than one and comprise and specific nucleic acid sequence complementary nucleotide sequence.Carrier itself can be used as mark and uses, and perhaps can comprise detectable marker or is modified by detectable marker, perhaps surveys oligonucleotide and can carry such mark.The carrier that is suitable for the inventive method includes but not limited to nanoparticle, quantum dot, dendrimer (dendrimers), semi-conductor, pearl, goes up conversion or enough suitable inorganic or organic molecule or its combinations of down-conversion fluorescent powder (up-or down-convertingphosphors), macro-molecular protein, grease, carbohydrate or virtually any size.

Used herein " surveying oligonucleotide (detector oligonucleotide) " or " surveying oligonucleotide (detection oligonucleotide) " are oligonucleotide thus defined: its comprise can be with the complementary nucleotide sequence or the assignment of genes gene mapping (that is, in sample, hybridizing) nucleotide sequence on target nucleic acid molecules.Survey the suitable of oligonucleotide but nonrestrictive example comprises DNA, RNA, PNA, LNA or its combination.Survey oligonucleotide and can comprise native sequences or composition sequence, have or do not have the Nucleotide of modification.

Term as used herein " mark " refers to pass through the detectable marker of photonics, electronics, electrooptics, magnetic, gravity, acoustics, zymetology or other physics or chemical means detection.Term " mark " refer to by as binding radioactivity mark nucleosides or with detectable marker be attached to oligonucleotide in conjunction with detectable marker.

" sample " used herein refers to comprise any amount of material nucleic acid, that can be used in the inventive method.For example, this sample can be biological sample or can obtain from the biological sample extraction that comes from people, animal, plant, fungi, yeast, bacterium, virus, tissue culture or viral cultures or its combination.They can comprise or extract from solid tissue's (for example marrow, lymphoglandula, brain, skin), body fluid (for example serum, blood, urine, saliva, seminal fluid or lymph liquid), bone tissue or individual cells.Alternatively, sample can comprise nucleic acid molecule purifying or partially purified and for example buffer reagent and/or reagent, this buffer reagent and/or reagent be used to obtain can successful implementation the inventive method felicity condition.

In one embodiment of the present of invention, the target nucleic acid molecules in the sample can comprise RNA, plasmid DNA, the nucleus of genomic dna, geneome RNA, expression or come from cell organelle (as plastosome) or parasitic nucleic acid, or its combination.

" the biological complex degree " of nucleic acid molecule used herein refers to be present in the Nucleotide number of the non-repeated nucleotide sequences in the nucleic acid molecule, for example, Lewin shows GENE EXPRESSION 2, SecondEdition:Eukaryotic Chromosomes, 1980, John Wiley ﹠amp; Sons described in the New York, is incorporated herein by reference.For example, its complexity of simple oligonucleotide that comprises 30 bases of non repetitive sequence is 30.Intestinal bacteria (E.coli) genome complexity that contains 4,200,000 base pair is 4,200,000, because it does not have tumor-necrosis factor glycoproteins basically.Yet human genome has similar 3,000,000,000 base pair, wherein much is tumor-necrosis factor glycoproteins (for example about 2,000,000,000 base pair).Total complexity of human genome (that is non-repetition nucleosides number) is 1,000,000,000 similarly.

The complexity of nucleic acid molecule such as dna molecular does not rely on the number (that is, be present in the nucleic acid molecule each not homotactic copy number) of different tumor-necrosis factor glycoproteinss.For example, if a DNA has the sequence of 1 long a Nucleotide, the sequence of long b Nucleotide of 5 copies, and the sequence of long c Nucleotide of 50 copies, its complexity is a+b+c, the repetition rate of sequence a is 1, the repetition rate of sequence b is 5, and the repetition rate of sequence c is 10.

Can be by calculating the Cot of DNA _1/2Determine not homotactic total length among the given DNA with laboratory facilities, can be by following formulate,

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

Wherein C is that single stranded DNA is at time t _1/2The time concentration (having reacted 1/2 o'clock), k is a velocity constant.Cot _1/2Required numerical value during two half renaturation of complementary strand of representation DNA.Normally, represent that with the Cot curve mode renaturation of DNA, Cot curve plotting have gone out to keep the mark (fraction) of DNA of strand (C/Co) with respect to the mark (1-C/Co) of the figure of the denary logarithm (log) of Cot or the renaturation DNA figure with respect to the denary logarithm of Cot.The Cot curve proposes (1968, Science 161:529-540) by Britten and Kohne in nineteen sixty-eight.The Cot curve display, the concentration of the sequence of every renaturation has determined the renaturation yield of given DNA.In contrast, Cot _1/2Representative is present in the not homotactic total length in the reaction.

The Cot of DNA _1/2Proportional with its complexity.Therefore, can pass through its Cot _1/2Cot with the standard DNA of known complexity _1/2Determining of DNA complexity finished in contrast.Usually, be used for determining that the standard DNA of DNA complexity is an e. coli dna, it has and its genome length (4.2 * 10 ⁶Individual base pair) Yi Zhi complexity is because each sequence in the bacillus coli gene group all is considered to unique.Therefore, following formula can be used for determining the biological complex degree of DNA.

In certain embodiments, the invention provides reliable detection and differentiation (promptly, identify) have among total people DNA coding mutation (as, the method of target nucleic acid molecules single nucleotide polymorphism), this method do not need at first preferably concrete dna sequence dna to be increased to carry out the zymetology reduced complexity by PCR or any other method.Particularly, method of the present invention comprises hybridization conditions combination (comprising reaction volume, salt, methane amide, temperature and test mode), the capture oligo sequence that is incorporated into substrate, exploratory probe and enough sensitive target nucleic acid molecules detection meanss, and wherein target nucleic acid molecules is discerned by capture oligo and exploratory probe simultaneously.

As shown among the embodiment, the present invention provides the method that successfully detects single nucleotide polymorphism in total human DNA by a step hybrid method first, amplification that this method does not need early stage or reduced complexity are with the selective enrichment target sequence, also without any need for the help of enzyme reaction, this step hybrid method comprises two hybridisation events: the first part of target sequence and the hybridization of capture probe, and the hybridization of the second section of described target sequence and exploratory probe.Fig. 1 shows a step hybrid method synoptic diagram.As discussed above, two hybridisation events all occur in the same reaction.The target thing can at first be combined on the capture oligo, and then hybridizes with the nanoparticle of exploratory probe as shown in synoptic diagram, and perhaps the target thing can at first combine with exploratory probe, and then hybridizes with capture oligo.

In another embodiment, the invention provides and detect and distinguish the method that has the target nucleic acid molecules of one or more discontinuous nucleosides sudden changes among (promptly identifying) total DNA reliably, this method does not need at first preferably specific dna sequence dna to be increased to carry out the zymetology reduced complexity by PCR or any other method.For example, method of the present invention can be used for distinguishing two or more target nucleic acid molecules from two or more different plant species of same genus, wherein these species have the difference of two or more discontinuous kernel thuja acids, and the detection oligonucleotide that can use the capture oligo with one or more nucleotide differences and/or have one or more nucleotide differences is distinguished.Method of the present invention also can be used for distinguishing two or more species that two or more continuous nucleotide differences are arranged in the same genus.

In one embodiment, can use two step hybrid methods to implement method of the present invention.Fig. 2 shows two step hybrid method synoptic diagram.In this method, hybridisation events occurs in two independent reactions.The target thing at first is combined on the capture oligo, remove all non-binding nucleic acid after, carry out second time hybridization, hybridization for the second time provides the exploratory probe that can be incorporated into the second section of captured object nucleic acid specifically.

The method of the present invention that relates to two step hybrid methods is during the hybridisation events first time (being catching of target nucleic acid molecules), need not to give some unique compliance characteristics of exploratory probe (such as the quick fusion behavior of high Tm and nanoparticle probe) can implement, because reaction takes place in two steps.First step does not have strict with the degree that is enough to only catch required target sequence.Therefore, provide second step (combination of exploratory probe) to obtain required specificity at target nucleic acid molecules.The combination of these two identity hybridisation events allows all specificitys at target nucleic acid molecules.Yet in order to obtain this sharp specificity, selected hybridization conditions is strict.Under such stringent condition, have only a spot of target thing and the exploratory probe probe that is hunted down to catch.The usually very little so that fluorescent method of standard of the amount of target thing can not detect, because it is buried in the background.Therefore, for purposes of the invention, it is very important using the exploratory probe that suitably designs to detect these a spot of target things.Exploratory probe described in the invention is present in carrier part, is modified to usually to comprise many detection oligonucleotide, makes the hybridization kinetics of this exploratory probe strengthen like this.The second, exploratory probe is also with one or more highly sensitive labeling section mark of assigning to, and these highly sensitive mark parts allow the very little acquisition target thing-exploratory probe complex body of amount detection with suitable testing tool.Therefore,, use highly sensitive detection system simultaneously, just make this method to implement exactly because suitably adjusted all factors.

Two step hybrid methods of the present invention can comprise any exploratory probe described herein is used to detect step.In preferred embodiments, the nanoparticle probe is used for second step of present method.Identical in the place of using the nanoparticle probe in second hybridization step and stringency and the first step, the detection oligonucleotide on the nanoparticle probe can be longer than capture oligo.Like this, the necessary condition of the specific characteristic of nanoparticle probe (high Tm and fusion behavior fast) has just no longer needed.

Capture oligo and exploratory probe that one step and two step hybrid methods suitably design in the present invention provide the method with respect to target nucleic acid sequence in the former test sample, new, unexpected advantage.Especially, method of the present invention does not need amplification step, the purpose of this amplification step is with hit thing quantity maximization of sample, the relative concentration that reduces non-target sequence simultaneously is to increase the possibility in conjunction with the target thing, the amplification step as the detection method based on polymerase chain reaction (PCR) just needs.The specific detection that need not target sequence amplification in early stage provides huge favourable condition.For example, amplification often causes studying or the pollution of diagnostic test chamber, causes false positive test results.PCR or other target amplification need the personnel of special training, the enzyme of costliness and special equipment.The most important thing is that the efficient of amplification can be with each target sequence and primer to changing, occur wrong even failure when causing determining to be present in the relative quantity of target sequence in the genome or target sequence.In addition, method of the present invention relates to step seldom, and thereby than based on the nucleic acid target material detection method of gel, as Southern and Northern trace testing method, be more prone to implement also more effective.

In one embodiment, the invention provides the method for target nucleic acid sequence in the test sample, wherein sample comprises the nucleic acid molecule of biological complex degree than the biological complex Du Genggao of the nucleic acid molecule of amplification, target nucleic acid sequence and known nucleotide sequence have the difference of a Nucleotide at least, the method comprising the steps of: a) provide the addressable that is combined with capture oligo substrate, wherein at least a portion of the first part that capture oligo can the recognition objective nucleotide sequence; B) provide and comprise the exploratory probe of surveying oligonucleotide, wherein survey oligonucleotide can with at least a portion hybridization of the second section of target nucleic acid sequence in the step (a); C) capture oligo can be specifically, optionally effectively hybridize with the first part of target nucleic acid sequence, and exploratory probe can be specifically, optionally with the second section of target nucleic acid sequence effectively under the condition of hybridization, sample is contacted with exploratory probe with substrate; Whether and d) detect capture oligo and exploratory probe hybridizes with the first part and the second section of target nucleic acid sequence.In another embodiment, be combined with many capture oligos on the addressable substrate, a plurality of parts that can the recognition objective nucleotide sequence, comprising one or more exploratory probes of surveying oligonucleotide can hybridize with one or more parts of target nucleic acid sequence, and the oligonucleotide that can not be hunted down is discerned.

In another embodiment, the invention provides the method for identifying single nucleotide polymorphism in the sample, wherein sample comprises the nucleic acid molecule of biological complex degree than the biological complex Du Genggao of the nucleic acid molecule of amplification.The method comprising the steps of: the addressable that is combined with at least one capture oligo substrate a) is provided, and wherein said at least one capture oligo can be discerned the nucleic acid target material that comprises specific polymorphism; B) provide and be combined with the exploratory probe of surveying oligonucleotide, wherein survey oligonucleotide can with at least a portion hybridization of the nucleic acid target material of step (a); C) capture oligo can be specifically, optionally effectively hybridize with nucleic acid target material, and exploratory probe can be specifically, optionally with nucleic acid target material effectively under the condition of hybridization, sample is contacted with exploratory probe with substrate; Whether and d) detect capture oligo and exploratory probe hybridizes with nucleic acid target material.In another embodiment, the addressable substrate is combined with many capture oligos, a plurality of parts that can the recognition objective nucleotide sequence, comprise survey oligonucleotide exploratory probe can with the part hybridization of target nucleic acid sequence, and the oligonucleotide that is not hunted down is discerned.

Method of the present invention can be distinguished two sequences that a nucleotide difference is only arranged.Therefore, in specific embodiment, method of the present invention can be used for detecting the objectives nucleic acid molecule with at least one coding mutation.In preferred embodiments, this sudden change is single nucleotide polymorphism (SNP).

In another embodiment, surveying oligonucleotide can be by detectable ground mark.The different methods of mark polynucleotide is well known in the prior art, can be conveniently used in the method described herein.In certain embodiments, detectable marker of the present invention can be fluorescence, luminous, Raman (Raman) is active, phosphorescent, radioactive or in scattered light effectively, mark with unique quality, or other the specific detectable physics or the mark of chemical property with some other easy detections, in order to strengthen described detectable characteristic, this mark can be assembled or one or more copy is attached on the carrier, such as dendrimer (dendrimer), molecule aggregates, quantum dot or pearl.Mark allows to survey by for example photonics, electronics, acoustics, acoustooptics, gravity, electrochemistry, zymetology, chemistry, Raman (Raman) or mass spectrum means.

In one embodiment, exploratory probe of the present invention can be to be combined with the nanoparticle probe of surveying oligonucleotide.Because come from the unique physical chemical property of its size, nanoparticle becomes very interested research object.Because these characteristics, nanoparticle for more responsive than traditional detection method, specificity is stronger, more have the development of the new bio transmitter of cost advantage that very promising approach is provided.The method of synthesizing nano-particle and research by the methodology of its deutero-characteristic in the past 10 obtained development (Klabunde, editor, Nanoscale Materials in Chemistry, WileyInterscience, 2001) widely.Yet, because inherent uncompatibility between nanoparticle and these two kinds of totally different materials of biomolecules, thereby lack strong method, and so cause the application of nanoparticle on biological significance to be restricted with interested biomolecules functionalized nano particle.Developed the high efficiency method that with the oligonucleotide functionalized nano particle of modifying.See U.S.Patent No.6,361,944 and 6,417,340 (assignee:Nanosphere, Inc.), with its integral body in the lump as a reference.This method obtains the nanoparticle by oligonucleotide height functionalization, and it has surprising particle stability and hybridization characteristic.Particle by the dna modification of its stability of solution susceptible of proof gained is very strong, wherein stability of solution comprise the raising of electrolysis concentration, to centrifugal or refrigerated is stable and thermostability when repeating heating and cooling.This stowage is that may command is revisable.The nanoparticle of different sizes of functionalization and composition, and on nanoparticle, load the oligonucleotide recognition sequence and can control by stowage.Suitable but example nonrestrictive nanoparticle comprises U.S. patent No.6,506,564, international patent application No.PCT/US02/16382, be filed in the U.S. patent application No.10/431 on May 7th, 2003,341, and those nanoparticles described in the international patent application No.PCT/US03/14100, integral body is in the lump as a reference herein for all these documents.

The stowage of the golden nanometer particle probe of the nanoparticle of aforesaid preparation dna modification, especially dna modification has made at the scheme on the new colorimetric meaning of oligonucleotide developed.This method is based on the hybridization of two different zones of two golden nanometer particle probes and interested DNA target thing.Because each probe is by a plurality of oligonucleotide functionalization with identical sequence, thereby when having enough target things, the combination of target thing causes the formation of target dna/golden nanometer particle probe aggregate.Since spacing of particle from shortening, the identification of DNA target thing causes colorimetric to change.This colorimetric changes and can monitor or use the bore hole visual monitoring by optical instrument with the UV-vis spectrophotometer.In addition, color burn when solution concentration is to film.Therefore, simple colorimetric changes provides the evidence that has or do not exist specific dna sequence.Use this test, can detect model DNA target thing that flies mole (femtomole) amount and nanomolar concentration and the nucleotide sequence that polymerase chain reaction (PCR) increases.Importantly, Au probe/DNA target thing complex body has shown that fusion changes very fast, makes it to become the DNA target substance markers of high special.In model system, the insertion of a base, deletion or mispairing can be by monitoring that based on the spot test (spot test) of C﹠T or by spectrophotometry the fusion transformation of aggregate detects (Storhoff etc. at an easy rate, J.Am.Chem.Soc., 120,1959 (1998)).Also can referring to, for example, U.S.Patent No.5,506,564.

Because rapidly fusion changes, when hybridization with detect under very strict condition (for example, the melting temperature (Tm) than the probe/target thing of intact coupling is low once) and when carrying out,, also can detect the intact target thing of coupling even there is the target thing of mispairing.Being accompanied by the wideer fusion that for example observes by molecular fluorescence group mark changes, with the approaching temperature condition of melting temperature (Tm) under hybridization and detect and will cause tangible Loss Of Signal, because the fusion of the part of probe/target thing complex body causes lower sensitivity, owing to the influence of mismatch probe signal, the part hybridization of mismatch probe/target thing complex body also causes lower specificity simultaneously.Therefore, the nanoparticle probe provides detection specificity higher nucleic acid detection method.

As described here, nanoparticle probe, especially golden nanometer particle probe, astonishing and direct SNP that be suitable for genomic dna unexpectedly detects, and need not amplification.At first, the fusion very fast that observes in nanoparticle oligonucleotide exploratory probe is converted into has unprecedented, surprising detection specificity, even also can allow distinguishing of single base under the human genome background.The second, in test, can further provide the sensitivity lifting of superelevation based on the method for amplifying signal of silver based on dna microarray.

For example can use optics or flat bed scanner to detect nanoparticle in the methods of the invention.Scanner can be connected on the computer, has loaded the software that can calculate gray-scale value on the computer, and the gray-scale value that calculates provides the quantitative values of the nucleic acid amount that is detected.

Suitable scanner comprise those be used for file scanning to computer, can be (for example with the scanner of reflection mode (for example flat bed scanner) work, other standard scanner that can carry out the responsive determinator of gray scale this function or that use identical optics device, any kind and be used to scan substrate of the present invention through repacking, the flat bed scanner that comprises substrate holder through repacking) (present, as to find to use scanner as yet) with transmission mode work.The resolving power of scanner must be enough big, so that the reaction zone on the substrate is greater than the single pixel of scanner.But under the prerequisite that the change detected that produces in test can be observed with respect to substrate (for example, the greyness that produces during silver dyes can observe under white background, but can not observe under gray background), scanner can use with any substrate.Scanner can be black and white scanner or preferred color scanner.

Most preferably, described scanner is the standard color scanner type that is used for file scanning is gone into computer.Such scanner low price, commercial easy acquisition.For example, can use EpsonExpression 636 (600 * 600dpi), UMAX Astra 1200 (300 * 300dpi) or Microtec 1600 (1600 * 1600dpi).Scanner is connected on the computer, and the software of the image of handling the scanning substrate acquisition is housed in the computer.This software can be the standard software of commercial easy acquisition, as Adobe Photoshop 5.2 and Corel Photopaint 8.0.Use these computed in software gray-scale values that the means that quantize test result are provided.

These softwares also can add colorful digital to coloured speckle, the image that can obtain scanning (as, printout), check that these images can obtain the qualitative test result of nucleic acid existence, the amount of nucleic acid, perhaps both all have.Also find in addition, can be by from the color of representing positive findings, deducting the sensitivity that the color of representing negative findings improves test.

Computer can be a standard personal computer, and it is in commercial easy acquisition.Like this, when on substrate, testing, use the standard scanner be connected in the standard computer that standard software is housed, convenience, easy, cheap detection and the means of quantitative nucleic acid are provided.Scanning (image) also can be stored in the computer, keeps outcome record, so that further reference or use.Certainly, if necessary, can use complicated apparatus and software more.

Can use silver to dye with the catalysis silver reductive nanoparticle of any kind.Noble metal (as, the nanoparticle of Jin Heyin) making is preferred.See Bassell etc., J.Cell Biol., 126,863-876 (1994); Braun-Howland etc., Biotechniques, 13,928-931 (1992).If be used for the reduction of not catalysis of the nanoparticle silver of detection of nucleic acids, silver ions can be combined catalytic reduction with nucleic acid so.See Braun etc., Nature, 391,775 (1998).Also have, known silver dyes the district and can react with the phosphate group on the nucleic acid.

Silver dyes and can be used for producing or strengthen detectable variation in any test of carrying out on the substrate, comprise above-described those.Particularly, have been found that silver dyes the sensitivity of the test that has greatly improved use single type nanoparticle, so that usually can not use nanoparticle layers, polymerization probe layer and core probe layer.

In another embodiment, the oligonucleotide that is attached to substrate can place between two electrodes, and nanoparticle can be made by electro-conductive material, and step in the inventive method (d) can comprise the variation that detects electroconductibility.In another embodiment, many oligonucleotide in the array of spots are attached on the substrate, each oligonucleotide wherein can be discerned the different target nucleotide sequence, each oligonucleotide spot is all between two electrodes, nanoparticle is made by electro-conductive material, and step in the inventive method (d) comprises the variation that detects electroconductibility.Electrode can be made by for example gold, and nanoparticle also is made of gold.Alternatively, substrate can dye to distinguish to contact to produce electroconductibility and change with silver.

In specific embodiment, the nucleic acid molecule in the sample has the biological complex degree that is higher than amplifier nucleic acid molecule.Those skilled in the art use as Lewin, GENE EXPRESSION 2, SecondEdition:Eukaryotic Chromosomes, 1980, John Wiley ﹠amp; Method described in the Sons, New York can be measured the biological complex degree of target nucleic acid sequence at an easy rate, and the document herein in the lump as a reference.

Hybridization kinetics definitely depends on co-reactant (reaction partners) the i.e. concentration of the chain of necessary hybridization.In the DNA of cell sample, the amount of the DNA of total genome, plastosome (if present) and extrachromosomal element (if present) only is several micrograms in the extraction of specified rate.Therefore, the actual concentrations of the co-reactant that hybridize will depend on the size of these co-reactants and the complexity of the DNA that is extracted.For example, when comparing the DNA sample of the different complexities of different sources, exist the target sequence of 30 bases of a copy to have different concentration in each genome.For example, approximately low 1000 times of the concentration of the concentration ratio of same target sequence in the total human DNA of 1 microgram in 1 microgram DNA of bacteria are hanged down about 1,000,000 times than the content that is present in the 1 microgram miniplasmids DNA sample.

The high complexity (1 * 10 of human genome ⁹Individual Nucleotide) require unusual high specificity, because redundancy and similar sequence are arranged in the genomic dna.For example, in order from whole human genomes, to distinguish the chain of catching, need have 40,000, the degrees of specificity of 000: 1 separating capacity with 25 poly-oligonucleotide.In addition, because in 25 poly-acquisition sequences, wild-type and mutant target thing only have the difference of a base, thereby, in order successfully to carry out gene type (genotyping), need to distinguish two target things with 96% homology.That the present invention surprisingly, unexpectedly provides is effective, special, detect with the nucleic acid molecule that increases delicately and compare, and has the method for the target nucleic acid molecules of higher complexity.

The biological complex degree situation that comes from target nucleic acid molecules in the sample of human tissue is 1,000,000,000, but may be higher or lower than 10 times of plant or animal gene groups.Preferably, the biological complex degree is about 50,000 to 5,000,000,000.Most preferably, the biological complex degree is about 1,000,000,000.

In one embodiment, hybridization conditions is effective for specificity, the selective cross of capture oligo and/or detection oligonucleotide and target nucleic acid sequence, under such hybridization conditions, can detect the mispairing of single base, even at described target nucleic acid is to have 50, under the situation of the part of the nucleic acid samples of 000 or 50,000 above biological complex degree, shown among for example following embodiment.

Method of the present invention can be further used for identifying concrete biology microbial species (as staphylococcus) and/or be used to detect the gene (for example, giving the mec A gene of microbiotic X-1497 resistance) of giving antibiotics resistance.

X-1497 resistance staphylococcus aureus strains (MRSA) has become primary nosocomial pathogens in the world wide.In the large-scale teaching hospital of the U.S., relevant with these bacteriums in the staphylococcal infections in all hospital sources above 40%.Recently they have been popular in more small-sized hospital (have incidence is 20% in the hospital of 200 to 500 berths), the family that nurses a baby in addition (Wenzel etc., 1992, Am.J.Med.91 (Supp 3B): 221-7).The MRSA bacterial strain is uncommon also to be that the most unfortunate characteristic is the ability that they obtain extra resistance factor, and described resistance factor can suppress these bacterial strains other are used for the antibiotic susceptibility of chemotherapy.Now, such multiresistance bacterial strain is worldwide popular, " advanced person " form of this pathogenic agent carry resistance mechanism to most of available antiseptic-germicides (Blumberg etc., 1991, J.Inf.Disease, Vol.63, pp.1279-85).

The main genetic constitution of X-1497 resistance is so-called mec A gene.This gene is found on the dna fragmentation of non-staphylococcus origin one section the unknown, is likely by ancestors MRSA cell to obtain from external source.Penicillin (penicillin) conjugated protein (PBP) (Murakami and the Tomasz of mec A genes encoding PBP2A by name, 1989, J.Bacteriol.Vol.171, pp.874-79), this albumen has low-down affinity to the man family antibiotic of whole beta lactan.At present, PBP2A is a class " substituting " cell walls synthetic enzyme, can be at normal PBP (agent of cell walls synthetic normal catalytic) fill-in because the beta beta-lactam antibiotics in the environment and complete deactivation again during functionating, are taken over the synthetic task of vital cell walls in the staphylococcus.Early stage transposon inactivation experiment confirm the important attribute of mecA gene and its antibiotics resistance phenotype genes product P BP2A, in this experiment, transposon Tn551 changes mec A gene over to.Result of experiment is, minimum inhibition concentration (MIC) the value 1600 μ g/mls of resistance level from the parent bacterium significantly drop to low value (Matthews and the Tomasz of about 4 μ g/ml in the transposon mutant body, 1990, Antimicrobial Agents and Chemotherapy, Vol.34, pp.1777-9).

Along with the increase of antibiotics resistance strain system, and the increase of the infection number that causes by the coagulase negative staphylococcus kind, the staphylococcal infections in the hospital becomes and is difficult to treatment more.Be used for determining that owing to many species (species form and analyze) and the test of antibiotics resistance need long time, effective methods of treatment of these infection has reduced.The Rapid identification that species and antibiotics resistance situation have been arranged just can more early be implemented patient's treatment process, and less use Broad spectrum antibiotics.Therefore, need obviously apace, highly delicately, optionally identify and distinguish the method for staphylococcus species and/or detect the method for mec A gene.

In another embodiment, the invention provides oligonucleotide sequence and its antisense complementary sequence of being used for staphylococcus species formation analysis and/or X-1497 resistant gene (mec A) detection, and the nanometer particle to mark probe, method and the test kit that use these sequences.The sequence of these designs has the sensitivity and the selectivity of height to staphylococcus kind or mec A gene, and wherein mec A gene produces the antibiotics resistance of some form.These sequences can be used for mec A gene test or the staphylococcus species form the predetermined purpose of analyzing, and also can be used as the negative control of other system.At present, can as shown in following embodiment, arbitrary probe groups among Tuf3 and Tuf4 or Tuf5 and the Tuf6 be used for streptococcus aureus and staphylococcus epidermidis are distinguished with probe Tuf 2.The 16S of sequence mark is used for detecting 16S rRNA that Staphylococcus comprises or the existence of DNA.Can be used for preparing these sequences as capture probe and/or nanometer particle to mark probe as traditional methods such as standard phosphoramidite chemical methods.

In another embodiment of the invention, these sequences can be used for using the staphylococcus species of non-amplifying genom DNA to form in analysis and/or the mec A detection method.Mec A gene order of the present invention has been used under current test condition and mode, detects double-stranded PCR production concentration and is low to moderate 1 * 10 ^-13M (100fM, 3 * 10 ⁶Individual copy), ultrasonic degradation total genomic dna content is few to 33ng (1 * 10 ⁷Individual copy) the mec A gene (seeing Figure 21) in the 50 μ l reaction systems.Use pcr amplification gene product or total bacterial genomes DNA have also detected the sensitivity and the specificity of the Tuf gene order that is used for streptococcus aureus and staphylococcus epidermidis species formation analysis in the test.The lower detection boundary of determining is in the 50 μ l reaction systems at present, double-stranded PCR product 1 * 10 ^-12M (1pM, perhaps 3 * 10 ⁷Individual copy), ultrasonic degradation genomic dna 150ng (5 * 10 ⁷Individual copy) (sees Figure 20).The condition of implementing these tests is described below.Method of the present invention is used the bacterial genomes DNA of reduced complexity or the target amplification of premenstruum not, have the dna sequence dna of one or more base differences by differentiation, provide surprisingly effectively, sensitive, detect the method for different staphylococcus species specifically.

In another one embodiment of the present invention, when using the pcr amplification period of the day from 11 p.m. to 1 a.m, can use second nanoparticle probe to replace as PCT/US01/46418 (Nanosphere, Inc., Assignee) acquisition sequence that is attached to array substrate described in, its integral body are in the lump as a reference.When target dna and two nanoparticle probe hybridizations caused colour-change, this system can detect (for example color or scattering of light) by optical means.Such test can be used for the staphylococcus species described in the above-mentioned test and identifies or mec A gene test purpose.

Embodiment

To further set forth the present invention by following illustrative embodiment.Provide these embodiment not really want to limit by any way the present invention by way of example.In these embodiments, all per-cent is if solid then is weight percentage, if liquid then is volume percent, if not explanation, all temperature are degree centigrade.

Embodiment 1

Use the nanoparticle probe in non-amplifying genom DNA, to identify a step hybrid method and the two step hybrid methods of SNP

With the PCT/US97/12783 that is filed on July 21st, 1997, be filed on June 26th, 2000 PCT/US00/17507, be filed in the golden nanometer particle oligonucleotide probe that the method preparation of describing among the PCT/US01/01190 in January 12 calendar year 2001 is used to detect target factor II, MTHFR and factor V sequence, these document integral body are in the lump as a reference.Fig. 3 uses the dna microarray with wild-type or mutant capture probe oligonucleotide from conceptive the elaboration, the golden nanometer particle probe that is combined with oligonucleotide is used to detect the purposes of target dna.Be incorporated into the part complementation of the oligonucleotide sequence and the target sequence of nanoparticle, and be incorporated into the capture oligo sequence of glass-chip and another part complementation of target sequence.Under hybridization conditions, nanoparticle probe, capture probe and target sequence are combined together to form complex body.Can dye the detection signal that strengthens the gained complex body with conventional silver.

(a) preparation of golden nanometer particle

As Frens, 1973, Nature Phys.Sci., 241:20 and Grabar, 1995, Anal.Chem.67:735 is described, and gold colloid (diameter 13nm) is by using Citrate trianion reduction HAuCl ₄Prepare.Briefly, all glass waress are all cleaned in chloroazotic acid (3 parts of HCl, 1 part of HNO3), use Nanopure H ₂The O flushing is used oven drying before using then.HAuCl ₄With Trisodium Citrate available from Aldrich pharmaceutical chemicals company (Aldrich Chemical Company).Hydration HAuCl ₄(1mM 500mL) is used for stirring backflow.Then, add 38.8mM Trisodium Citrate (50mL) fast.Solution colour is from the light yellow red-purple (burgundy) that becomes, and refluxing continues 15 minutes.After being cooled to room temperature, use the 1 micron filter filter red solution of MicronSeparations Inc..Use Hewlett Packard 8452A type diode array spectrophotometer with ultraviolet-visible (UV-vis) spectroscopic method, use Hitachi8100 type transmission electron microscope to determine the feature of gold colloid with transmission electron microscopy (TEM) simultaneously.When assembling with target thing with 10-35 Nucleotide scope and probe oligonucleotides sequence, the gold particle of diameter 15nm can produce the visible colour-change.

(b) oligonucleotide is synthetic

With ABI 8909DNA synthesizer, under the single-column pattern, adopt phosphoramidite chemical method [Eckstein, F. (ed.) Oligonucleotides and Analogues:A Practical Approach (IRL Press, Oxford, 1991)], synthetic 1 micromole amount with MTHFR, factor II or factor V dna sequencing fragment complementary capture probe oligonucleotide.Acquisition sequence comprises any 3 '-amido modified dose, this 3 '-amido modified dose in array treating process as the adherent active group of covalency.By following standard DNA synthetic schemes synthetic oligonucleotide.Having 3 '-amido modified dose pillar, standard nucleotides phosphoramidite and reagent that is attached on the solid support thing obtains from Glen Research.In order to help purifying, last dimethoxytrityl (DMT) blocking group is not excised from oligonucleotide.After synthetic, use ammoniacal liquor that DNA cracking from the solid support thing is got off, the result is created in the dna molecular that 3 ' end comprises unhindered amina.With 0.03M Et ₃NH ⁺OAc ^-Damping fluid (TEAA), pH7, the 95%CH of 1%/min gradient ₃CN/5%TEAA carries out the reversed-phase HPLC chromatography with Agilent 1100 equipment series that reversed-phase column (vydac grain (Vydac)) is housed.Flow velocity 1mL/min, 260nm carry out UV and detect.After damping fluid is collected evaporation, with 80% acetic acid treatment 30min, DMT cracking from the oligonucleotide is fallen under the room temperature.Then that solution evaporation is extremely approaching dry, add entry, use ethyl acetate that cracked DMT is extracted from oligonucleotide aqueous solution.Measure the amount of oligonucleotide at 260nm absorbancy place, assess final purity by analyzing reversed-phase HPLC.

The acquisition sequence that uses in the test mthfr gene is as follows: MTHFR wild-type, 5 ' GATGAAATCG GCTCCCGCAGAC-NH ₂3 ' (MTHFR-SNP/Cap6-WT22; SEQID NO:1) and the MTHFR mutant, 5 ' ATGAAATCG ACTCCCGCAGACA-NH ₂3 ' (MTHFR-SNP/Cap7-mut22; SEQ ID NO:2).As follows with the corresponding capture oligo of factor V gene: factor V wild-type, 5 ' TGG ACA GGC GAG GAA TAC AGGTAT-NH ₂3 ' (FV-Cap-WT24; SEQ ID NO:3) and factor V mutant, 5 ' CTG GACAGG CAA GGA ATA CAG GTA TT-NH ₂3 ' (FV-Cap-mut26; SEQ ID NO:4).Factor II wild-type: 5 ' CTCAGCGAGCCTCAATGCTCCC-NH ₂3 ' (FII-SNP/Cap1-WT22; SEQ ID NO:5) and factor II mutant, 5 ' CTCTCAGCAAGCCTCAATGCTCC-NH ₂3 ' (FII-SNP/Cap1-mut23; SEQ IDNO:6).

Be designed for the exploratory probe oligonucleotide that detects factor II, MTHFR and factor V gene and comprise the steroid disulfide linkage, 5 ' termination has recognition sequence.The sequence of 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, (FV-Pro 46 for 5 ' Epi-CCA CAGAAA ATG ATG CCC AGT GCT TAA CAA GAC CAT ACT ACA GTG A 3 '; SEQ ID NO:9).

Come the synthesising probing needle oligonucleotide according to described capture probe synthetic method through following modification.At first, use the upholder that has the suitable Nucleotide of representing 3 ' end recognition sequence to replace amido modified dose of pillar.Second, the phosphoramidite (phosphoramidite) of modification that use comprises steroid disulphide (steroid disulfide) with 5 ' end steroid ring-type disulphide (steroid-cyclic disulfide) imports Connection Step (referring to Letsinger etc., 2000, Bioconjugate Chem. 11: 289-291 and PCT/US01/01190 (Nanosphere, Inc.), these content whole are in the lump as a reference).Can prepare phosphoramidite by the following method: under dehydration conditions, with epiandrosterone (epiandrosterone) (0.5g), 1,2-dithiane-4, the p-methyl benzenesulfonic acid (p-toluenesulfonic acid) in 5-glycol (0.28g) and the toluene (30mL) be 7 hours (Dean Starkapparatus) of solution backflow (15mg); Remove toluene then under reduced pressure, raffinate places ethyl acetate.This solution with water washing, dried over sodium sulfate is condensed into the syrupy shape raffinate, with its standing over night in pentane/ether, obtain white solid, Rf value (ether is as extract for TLC, silicon chip) and be 0.5 steroid dithio ketal (steroid-dithioketal) compound (400mg); Contrast, the epiandrosterone and 1 that similarity condition obtains down, 2-dithiane-4, the Rf value of 5-glycol is respectively 0.4 and 0.3.Recrystallization obtains white powder, mp 110-112 ℃ from pentane/ether, ¹H NMR, δ 3.6 (1H, C ³OH), 3.54-3.39 (2H, the dithiane ring of m 2OCH), 3.2-3.0 (4H, m 2CH ₂S), 2.1-0.7 (29H, m steroid H); C ₂₃H ₃₆O ₃S ₂(M+H) mass spectrum (ES ⁺) calculated value is 425.2179, measured value is 425.2151.Anal. (C ₂₃H ₃₇O ₃S ₂) S: calculated value 15.12; Measured value 15.26.In order to prepare steroid disulphide ketal phosphoramidite derivative (steroid-disulfide ketal phosphoramidite derivative), steroid dithio ketal (100mg) is dissolved in THF (3mL), in the dry ice alcohol bath, cool off, add N, N-diisopropylethylamine (80 μ L) and β-cyanoethyl chlorine diisopropylphosphoramidite (β-cyanoethylchlorodiisopropylphosphoramidite) (80 μ L) continuously; Then, mixed solution is heated to room temperature, stirs 2 hours, and (100mL) mixes with ethyl acetate, with 5% hydration NaHCO ₃And water washing, use dried over sodium sulfate, be concentrated into drying.Residue places the methylene dichloride of minimum, and-70 ℃ add hexane and make it precipitation, vacuum-drying; Product 100mg; ³¹P NMR 146.02.DNA is synthetic finish after, under the ammoniacal liquor condition, the oligonucleotide that will connect epiandrosterone disulphide (epiandrosterone-disulfide) deprotection from the upholder is according to aforesaid method use reversed-phase column purifying on HPLC.

(c) oligonucleotide is attached on the golden nanometer particle

4 μ M oligonucleotide solution and～the golden nanometer particle colloidal solution of the 15nm citrate-stableization of 14nM final volume be in the solution of 2mL insulation earlier 24 hours with the preparation probe.Under the room temperature, the salt concn in the solution progressively was increased to 0.8M in 40 hours.Gained solution passes through 0.2 μ m cellulose acetate filter membrane, and 13,000G rotates 20 minutes granulation nanoparticle probes.After removing suspended substance, granulate is resuspended in the water.Step in the end with probe solution granulation once more, and is resuspended in probe storage damping fluid (10mM phos, 100mM NaCl, 0.01%w/v NaN ₃).Based on 520nm (ε=2.4 * 10 ⁸M-1cm ^-1) after the light absorption value estimated concentration, concentration is adjusted to 10nM.

The following special nanoparticle-oligonucleotide conjugate thing that is used for factor II, MTHFR and factor V DNA prepares 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 connector element by the preparation of epiandrosterone disulphide group; The number of n representative identification oligonucleotide.

(d) preparation of dna microarray

Use GMS417 matrix arrayer (Affymetrix) will catch chain alignment in Superaldehyde slide glass (Telechem) or CodeLinke slide glass (Amersham, Inc.) on.The layout design of arranging spot is carried out a plurality of hybridization for permission on each slide glass, such design can be by obtaining the method that slide glass is divided into independent test hole with silicone resin pad (Grace Biolabs).Wild-type and mutant become in the spot damping fluid three times to become spots what manufacturer provided.Carry out the arrangement aftertreatment of slide glass according to the scheme that manufacturer is recommended.

(e) hybridization

Factor V SNP detects testing method

Adopting following scheme to carry out factor V SNP detects.Genotype is people's placenta dna of the ultrasonic degradation of homozygous wildtype, and perhaps salmon sperm DNA (Sigma) is used ethanol sedimentation, is dissolved in 10nM FV probe solution.Add other compositions in mixture, final hybridization mixture (5 μ L) comprises 3xSSC, 0.03%Tween20,23% methane amide, 5nM FV probe and 10 μ g people DNA or reference explanation.After 99 ℃, 4 minutes the heat denatured step, hybridization mixture is added to the detection hole.Array is incubated 90 minutes in 50 ℃.Under the room temperature array immersed 0.5M NaNO ₃, 0.05% Tween 20 1 minutes, the flushing after beginning thus to hybridize.Remove pad, once more in 0.5M NaNO ₃Flushing detects slide glass in/0.05%Tween 20 solution, 3 minutes (2x) of insulation under the room temperature, mild agitation during insulation.As mentioned above, slide glass dyes with silver enhancement solution, and is dry on rotary drier, in Array Worx  biochip-reader (Model no.AWE, Applied Precision Inc., Issaquah, WA, U.S.A.) upward imaging.

(f) result

Factor V SNP detects

Fig. 4 is presented on the Superaldehyde slide glass, and the SNP of factor V gene distinguishes among the human genome DNA.Hot-wire array comprises wild-type and mutant is caught spot.Array that the top shows and wild-type human genome DNA hybridization, the array of bottom and the hybridization of the salmon sperm DNA of ultrasonic degradation.The signal of wild-type spot apparently higher than with the signal of the mutant spot of wild-type human genome DNA hybridization, show that it is and the homozygous wild gene type of factor V.Under hybridization conditions, do not observe the salmon sperm DNA hybridization signal, contrast as test with it.Also having carried out SNP with array on CodeLink  slide glass distinguishes.

Designed experiment shows that the hybridization that wild-type is caught on the spot is not owing to some other sequence, but is specific to the genome that comprises human factor V gene.Use the total DNA of human wild type, catch spot in wild-type and observed desired strong hybridization signal, and observed 3 times signal approximately weak at mutation spot.Yet, when extracting during as the target thing, does not observe signal, because this DNA does not comprise human factor V gene from the genomic dna of salmon essence.

Fig. 5 has shown for making this method can distinguish the importance of adjusting hybridization conditions in two target nucleic acids (this example is from the homozygous patient with factor V transgenation) with 1 Nucleotide (SNP site) difference.The proper equilibrium that must determine methane amide and SSC buffer salinity is so that target sequence preferentially combines its homology capture probe (for example Mut-A or Mut-B sequence).In addition, Fig. 5 has shown the influence of the capture oligo sequence of different sizes in the hybridization.Long 26 Nucleotide of Mut-A sequence, long 21 Nucleotide of Mut-B sequence.The result is presented at that specific signals has significant difference under the 15%FM/1XSSC condition, and does not have difference under the 25%FM/6XSSC condition, and two probes have all produced the strong signal with fine resolving ability.

In order to determine under similarity condition, whether can test sample in more than one SNP type, detected the existence of wild-type and mutant factor II and factor V gene in the genomic dna.Normal human subject (wt) genomic dna, be attached to capture oligo on the substrate and nanoparticle probe together, 40 ℃ were mixed among 35%FM and the 4X SSC 1 hour.Catch the preferential signal (Fig. 6) that produces in the spot in the wild-type of factor II and factor V gene.When usage factor II is that homozygous mutation type and factor V are when being the total genomic dna of individuality of homozygous wildtype, same array under the same hybridization conditions, catch spot and factor V wild-type in factor II mutant and catch spot and preferentially produce signal, clearly inerrably identified the genetic composition (Fig. 6) of this person on the SNP of these two genes configuration.This result shows, can design capture oligo sequence and hybridization conditions, makes under same hybridization conditions, can detect more than one SNP type in an array.And the SNP of wild-type and mutant DNA distinguishes it also is possible, and not rely on input DNA be normal source or sudden change source.

Two step hybrid methods

To the influence that SNP distinguishes, done more test in order to determine different stringent conditions.In test, use the methane amide of different weight percentage, make detection arrays under different stringent conditions, hybridize (Fig. 7).Be accompanied by the increase of strict degree, Loss Of Signal occurs, and this then the specificity of signal is got a promotion.In the contrast that does not have the target thing, almost do not observe signal.Measure the quantitative demonstration of signal in the spot, the signal of wild-type spot is than the high 3-6 of signal times (Fig. 7 B) of mutant spot.These results simultaneously for SNP in the genomic dna distinguish without any need for target amplification strategy (viewpoint) provide support.

Different lengths is comprised 20,21,24 or 26 Nucleotide (FV-WT20 (SEQ ID NO:13): 5 ' (GGACAGGCGAGGAATACAGG)-(PEG) x3-NH ₂, 3 ' FV-mut21 (SEQID NO:14): 5 ' (TGGACAGGCAAGGAATACAGG)-(PEG) x3-NH ₂3 ', FV-wt24 (SEQ ID NO:15): 5 ' TGG ACA GGC GAG GAA TAC AGG TAT-NH ₂3 ', FV-mut26 (SEQ ID NO:16): 5 ' CTG GAC AGG CAA GGA ATA CAG GTATT-NH ₂3 ') capture oligo be printed on as stated above on the CodeLink slide glass, add to 5 μ g normal people placenta genomic dnas (Sigma, St.Louis, MO) or factor V mutant human genomic dna (separate from storage culture GM14899, factor V defective, Coriell Institute).In the first step, place 20%FM, 30%FM or 40%FM and 4X SSC/0/04%Tween to be incubated 2 hours for 40 ℃ slide glass and DNA.Then, under the room temperature slide glass was washed in 2XSSC 3 minutes.After the flushing, the detection oligonucleotide of recognition factor V is added with the nanoparticle probe, then with 40 ℃ of insulations of mixture 1 hour.Silver dyes detection signal according to the method described above.The result shows that under the condition of adjusting through the best (being 30%FM in this example), people's wild-type DNA only produces signal on wild-type probe, and people's mutant DNA only produces signal (Fig. 8) at the sudden change capture probe.The strict degree of change condition causes the forfeiture (strict degree is low excessively) of resolving ability or the forfeiture (strict degree is too high) of signal.Fig. 9 shows the quantitative data of preferred (intermediary) hybridization conditions among Fig. 8.

Use DNA revision test under top condition of different concns then.As seeing among Figure 10, SNP distinguished it is successful when DNA concentration was 0.5 μ g, 1.0 μ g and 2.5 μ g.Therefore, this method can enough seldom total DNA detection SNP of people of amount (being less than 1 microgram).These results have also shown the capture oligo design, and stringent condition and catch the length of (and survey) probe and the suitable importance of coupling of nucleosides composition.

By carrying out 10 identical hybridization, measured the repeatability of two step hybrid methods at the different Kong Zhongyong of a slide glass 5 μ g wild-type complete genome group DNA.As shown in figure 11,10 separately in the hybridization standard deviation of the net signal intensity of coupling and mispairing not overlapping, show for each hybridization, can determine to import the SNP configuration of DNA reliably.

Next, detect the SNP of factor V, factor II and MTHFR and the wild type gene in the preparation same sample with this method.Under above-mentioned hybridization conditions, the complete genome group DNA with 5 μ g is incubated with the capture oligo of factor V, factor II or MTHFR.Add in second step and be specifically designed to the nanoparticle probe that detects factor V, factor II or MTHFR.The result of this test is shown in Figure 12-14, and it shows under the same terms, can analyze at least 3 heterogeneic SNP configurations simultaneously in single array.Figure 13 has shown the result that this polynary SNP detects in the patient DNA sample (GM16028), and this result is, all is heterozygosis for each gene.Figure 14 has shown the result that the polynary SNP of patient detects, and this patient's factor II is a heterozygosis, and factor V is a wild-type, and MTHFR is what suddenly change.This method has accurately been identified this patient's genotype (patient's sample GM00037).These results show that resolving ability is enough strong, can distinguish to isozygoty and the heterozygous mutant gene.For example, for any given SNP, a people can be wild-type, mutant or the heterozygous (i.e. a wild type gene and a mutated genes) of isozygotying.Three kinds of different situations of this of three independent SNP sites can correctly be identified respectively in independent once test.The result shows that method of the present invention can identify a plurality of SNP in the simple sample simultaneously.Yet only identified three SNP in this test, those skilled in the art can recognize that this only is a representative number.In with an array, can detect more SNP site.

Except these tests, two different experimenters also use the method described in these embodiment, respectively 8 different slide glasses are hybridized (DNA of patient GM14899 is 1 array of each slide glass, and the DNA of patient GM1600 is 2 arrays of each slide glass) with the DNA from 2 different patients.For the capture probe (mutant or wild-type) of each (factor II and the factor V) in 2 genes and each type, each array has 4 to repeat spot.Net signal intensity is average, classification begin to draw from lowest signal intensity then.For mispairing signal (every figure in than low value), standard deviation is added to average net signal three times.The signal of mutant and corresponding wild-type always is positioned at the top of one another.As shown in figure 15, even minimum strength of signal, the net signal of coupling also always adds that than the net signal of mispairing the triple standard deviation is big.Therefore, in each example, can both be to measure the correct configuration of SNP greater than 99% reliability.These results have further shown the powerful and repeatable of method described herein.

Embodiment 2

The hybridization conditions of the inventive method

Standard recommendation scheme (the T.Maniatis that is used for effective hybridization described in the prior, E.F.Fritsch, and J.Sambrook in " Molecular Cloning; A Laboratory Manual ", ColdSpring Harbor Laboratory, 1982, p324) mainly be that the regulation hybridization temperature is lower than Tm～10-20 degree centigrade, this Tm comprises salt and methane amide concentration according to selected hybridization conditions, calculates.Calculating the Tm value has diverse ways, and every kind of method is all based on oligonucleotide sequence and buffer conditions accurately.For example, can utilize computer program to carry out this calculating, these programs can be from commercial channels or online acquisition, for example developed and be kept at HYTHER on the Wayne state university website ^TMServer.The contriver is with HYTHER ^TMAll available programs on the server are used to carry out these calculating, have obtained the Tm value of capture probe and exploratory probe (being oligonucleotide).As shown in table 1, the Tm value of capture probe is lower than or is in close proximity to selects the temperature (promptly 40 ℃) be used to hybridize.Therefore, under these conditions, can expect that hybridization efficiency is very low.In addition, capture oligo directly is attached to substrate surface,, does not have catenation sequence that is, this means that the oligonucleotide of closest surface possibly can't participate in and the hybridization of target sequence, therefore further reduces effective Tm value.Instruction based on prior art, employed condition has unexpectedly obtained hybridization efficiently in the inventive method, especially at target sequence a very small fragment (for example 1/100 of the complex DNA mixture of human genome representative, 000,000 or 1million ' situation s%) under.

Table 1

		Use HyTher TMThe TM that (Wayne State University) calculates
					Do not have and revise	TM that done as follows and the surface bonding probe hybridization revises
		(35％FM)	Santalucia etc. (35%FM)	Fotin etc. (35%FM)
					Ravin	Sequence
FII- SNP/Cap1- wt22(SEQ ID NO：5)	CTCAGC GAGCCTCAATGCTCCC	46.7	37.0	45.0
					FII- SNP/Cap1- mut23	CTCTCAGC AAGCCTCAATGCTC C	47.2	35.7	46.3
MTHFR- SNP/Cap6- wt22(SEQ ID NO：6)	GATGAAATCG GCTCCCGCAGA C	40.3	35.5	43.0
					MTHFR- SNP/Cap7- wt22(SEQ ID NO：2)	ATGAAATCG ACTCCCGCAGAC A	40.7	36.2	44.0
FV-Cap- WT-24 (SEQ ID NO：15)	TGGACAGGCGAGGAATACAGGT AT	44.8	35.5	42.9
					FV-Cap- mut26 (SEQ ID N0：16)	CTGGACAGGCAAGGAATACAGG TATT	44.5	35.8	42.9
Probe
					FV-46 (SEQ ID NO：12)	5’Epi-CCA CAG AAA ATG ATG CCC AGT GCT TAA CAA GAC CAT ACT ACA GTG A 3’	54.8	49.2	57.6
FII-ProI-47 (SEQ ID NO：10)	5’Epi-TCC TGG AAC CAA TCC CGT GAA AGA ATT ATT TTT GTG TTT CTA AAA CT 3’	52.2	46.9	54.8
					MTHFR- Pro II-58 (SEQ ID NO：8)	5’Epi-AAA GAT CCC GGG GAC GAT GGG GCA AGT GAT GCC CAT GTC GGT GCA TGC CTT CAC AAA G 3’	68.4	58.6	68.5

Embodiment 3

The preparation of nanoparticle-oligonucleotide conjugate probe

In the present embodiment, prepared the representative nanoparticle-oligonucleotide conjugate exploratory probe that is used for mec A and Tuf gene target thing pcr amplification.Request for utilization in the PCT/US97/12783 on July 21st, 1997, apply on June 26th, 2000 PCT/US00/17507, apply for golden nanometer particle-oligonucleotide probe that method preparation described in the PCT/US01/01190 in January 12 calendar year 2001 is used to detect target mec A or Tuf gene order, these document integral body are in the lump as a reference.

(a) preparation of golden nanometer particle

As Frens, 1973, Nature Phys.Sci., 241: 20 and Grabar, 1995, Anal.Chem. 67: 735 is described, and gold colloid (diameter 13nm) is by using Citrate trianion reduction HAuCl ₄Prepare.Briefly, all glass waress are all at chloroazotic acid (3 parts of HCl, 1 part of HNO ₃) in clean, use Nanopure H ₂O flushing, oven drying before use then.HAuCl ₄With Trisodium Citrate available from Aldrich pharmaceutical chemicals company (Aldrich Chemical Company).Hydration HAuCl ₄(1mM 500mL) is used for stirring backflow.Then, add 38.8mM Trisodium Citrate (50mL) fast.Solution colour is from the light yellow red-purple (burgundy) that becomes, and refluxing continues 15 minutes.After being cooled to room temperature, use the 1 micron filter filter red solution of MicronSeparations Inc..Use Hewlett Packard 8452A type diode array spectrophotometer with ultraviolet-visible (UV-vis) spectroscopic method, use Hitachi8100 type transmission electron microscope to determine the feature of gold colloid with transmission electron microscopy (TEM) simultaneously.When assembling with target thing with 10-35 Nucleotide scope and probe oligonucleotides sequence, the gold particle of diameter 13nm can produce the visible colour-change.

(b) steroid disulphide is synthetic

With Milligene Expedite dna synthesizer, under the single-column pattern, adopt phosphoramidite chemical method [Eckstein, F. (ed.) Oligonucleotides and Analogues:A Practical Approach (IRL Press, Oxford, 1991)], synthetic 1 micromole amount with mecA and Tuf dna sequence dna complementary oligonucleotide.All solution is all available from Milligene (the synthetic level of DNA (DNA synthesisgrade)).Average joint efficiency from 98 to 99.8% does not wait, and is to help purifying, the cracking from the oligonucleotide of last dimethoxytrityl (DMT) blocking group is not fallen.

For ease of the hybridization of probe sequence and target thing, 5 ' of probe sequence holds the Desoxyadenosine oligonucleotide that comprises as transcribed spacer (except the Tuf2 probe has da ₁₀Peg, other all probes are da ₁₅Peg).

For produce 5 ' end steroid ring-type disulphide oligonucleotide derivative (see Letsinger etc., 2000, Bioconjugate Chem. 11: 289-291 and PCT/US01/01190 (Nanosphere, inc.) its disclosed content whole in the lump as a reference), the epiandrosterone phosphoramidite that connects with the ring-type dithiane carries out last ligation on the automatic DNA synthesizer DNA of Applied Biosystems, the epiandrosterone phosphoramidite that the ring-type dithiane connects is by 1,2-dithiane-4, the tosic acid in 5-glycol, epiandrosterone and the toluene (PTSA) makes.Phosphoramidite reagent can prepare as follows: epiandrosterone (0.5g), 1,2-dithiane-4, the solution of the tosic acid (15mg) in 5-glycol (0.28g) and the toluene (30mL) under dehydration conditions, reflux 7 hours (Dean Stark apparatus).Under reduced pressure, remove toluene then, raffinate is placed ethyl acetate.With this solution with water washing, dried over sodium sulfate, simmer down to syrupy shape raffinate, standing over night in pentane/ether obtains white solid steroid dithio ketal compound (400mg); Rf value (ether is as extract for TLC, silicon chip) is 0.5; Contrast, the epiandrosterone and 1 that similarity condition obtains down, 2-dithiane-4, the Rf value of 5-glycol is respectively 0.4 and 0.3.Recrystallization obtains white powder, mp 110-112 ℃ from pentane/ether, ¹H NMR, δ 3.6 (1H, C ³OH), 3.54-3.39 (2H, the dithiane ring of m2OCH), 3.2-3.0 (4H, m 2CH ₂S), 2.1-0.7 (29H, m steroid H); C ₂₃H ₃₆O ₃S ₂(M+H) mass spectrum (ES ⁺) calculated value is 425.2179, measured value is 425.2151.Anal. (C ₂₃H ₃₇O ₃S ₂) S: calculated value 15.12; Measured value 15.26.In order to prepare steroid disulphide ketone phosphoramidite derivative, steroid dithio ketal (100mg) is dissolved in THF (3mL), in the dry ice alcohol bath, cool off, add N continuously, N-diisopropylethylamine (80 μ L) and β-cyanoethyl chlorine diisopropylphosphoramidite (80 μ L); Then, mixed solution is heated to room temperature, stirs 2 hours, and (100mL) mixes with ethyl acetate, with 5% hydration NaHCO ₃And water washing, use dried over sodium sulfate, be concentrated into drying.Residue places the methylene dichloride of minimum, and-70 ℃ add hexane and make it precipitation, vacuum-drying; Product 100mg; ³¹P NMR 146.02.Under the situation of not removing last DMT, the oligonucleotide that synthetic epiandrosterone disulphide connects on AppliedBiosystems automatic gene synthesizer.After finishing, under the ammoniacal liquor condition, the oligonucleotide that epiandrosterone disulphide is connected deprotection from the upholder is with reversed-phase column purifying on HPLC.

Use 0.03M Et ₃NH ⁺OAc ^-Damping fluid (TEAA), pH 7, the 95%CH of 1%/min gradient ₃CN/5%TEAA carries out reversed-phase HPLC in the Dionex DX500 system that is equipped with Hewlett Packard ODS hypersil post (4.6 * 200mm, granular size is 5mm).Flow velocity is 1mL/min, carries out UV at the 260nm place and detects.Unmodified oligonucleotide with preparation HPLC purifying DMT protection.After damping fluid is collected evaporation, DMT cracking from the oligonucleotide is fallen with processing 30min under the 80% acetate room temperature.Solution evaporation near dry, is added entry, cracked DMT is extracted from oligonucleotide aqueous solution with ethyl acetate.Measure the amount of oligonucleotide at 260nm absorbancy place, with the final purity of reversed-phase HPLC assessment.

(c) microarray preparation

On dna synthesizer, synthesize the DNA that contains 3 '-amino and 5 '-amino according to following standard DNA synthetic schemes.By (citySunnyvale engraves on (printing) 1mM dna solution in StateCA), and amine-modified DNA is attached on the acetaldehyde microarry slides for Catalog no.MSP, CompanynameTelechem at ArrayIt damping fluid additive (plus).With Affymetrix GMS 417 matrix arrayers that microarray is directed on slide glass with 500 micron print needles.Microarry slides with acetaldehyde functionalized surface available from Telechem (catalog no.SMM, city Sunnyvale, stateCA).After engraving on, under the envrionment temperature, slide glass was put into moist cell 12-18 hour around.Remove slide glass and with its vacuum-drying 30 minutes to 2 hours.Then slide glass is washed twice in 0.2%w/v SDS, in water, wash twice to remove any residual non-binding DNA.Then, slide glass is placed the 2.5M sodium borohydride be dissolved in to contain the solution immersion treatment 5 minutes of 20%v/v 100% alcoholic acid 1X PBS.With 0.2%w/v SDS flushing three times, water washes twice, centrifugal drying then.

(d) oligonucleotide is attached to golden nanometer particle

Citrate-stable golden nanometer particle colloidal solution (about 10nM) and the sulfur modification-a for preparing according to the described method of B part according to the described method preparation of top A part _{15 peg}-probe oligonucleotides (4 μ M) is mixed, and under the room temperature it is left standstill in the scintillation vial of 20ml 6 hours.0.1M sodium hydrogen phosphate damping fluid and the 5.0M NaCl of pH 7.0 are added in the solution, obtain the solution of 0.01M sodium hydrogen phosphate and 0.1M NaCl, it was left standstill 16 hours again.Add sodium-chlor in the gradient mode, make NaCl concentration reach 0.8M in 36 hours, the solution of gained is incubated 18 hours again.Solution divided equally to 1ml eppendorf manage, in Eppendorf Centrifuge 5,414 14, centrifugal 25 minutes of 000rpm, obtain very shallow pink supernatant liquor, wherein having comprised the Radioactive colloidal gold (520nm place absorbance shown) of most oligonucleotide (260nm place absorbance is shown) together with 7-10%, is fine and close, black gel residue in the bottom of test tube.Remove supernatant liquor, resuspended residue in required water damping fluid.In the present embodiment, employed damping fluid comprises 0.1M NaCl, 10mM Trisodium Citrate and 0.01% sodium azide, and the pH value is 7.

Following nanoparticle-oligonucleotide exploratory probe prepares in this way with the amine-modified DNA capture probe that is specific to mecA and Tuf DNA: herein, oligonucleotide probe can perhaps be modified and be fixed on the gold grain as exploratory probe with the epiandrosterone connector with amine-modified and be fixed on the glass slide as capture probe.In other words, oligonucleotide and its reverse complementary sequence are interchangeable as capture probe or nanoparticle exploratory probe.

(a) exploratory probe

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Probe mecA 2:5 '-[amine-peg ₃-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-peg ₃-gcacttgtaagcacaccttcat-3 '] n (SEQID NO:44)

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

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

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

S ' expression is via the connector element of epiandrosterone disulphide group preparation; N represents the oligonucleotide of the different numbers that use in nanoparticle-oligonucleotide conjugate thing preparation.

Table 2

Title	SEQ ID NO：	Sequence 5 '-3 '	The staphylococcus bacterial classification
				Tuf 1	17 48	TFCTATTTCCGTACTACTGAC GTCAGTAGTACGGAAATAGAA (reverse complemental)	General T uf gene
Tuf 2	18 49	TTCTATTTCCGTACTACTGACGTAACT AGTTACGTCAGTAGTACGGAAATAGAA (reverse complemental)	General T uf gene
				Tuf 3	19 50	CCATTCTTCTCAAACTATCGT ACGATAGTTTGAGAAGAATGG (reverse complemental)	Streptococcus aureus
Tuf 4	20 5l	CCATTCTTCACTAACTATCGC GCGATAGTTAGTGAAGAATGG (reverse complemental)	Staphylococcus epidermidis
				Tuf 5	2l 52	CACACTCCATTCTTCTCAAACT AGTTTGAGAAGAATGGAGTGTG (reverse complemental)	Streptococcus aureus
Tuf 6	22 53	CACACTCCATTCTTCACTAACT AGTTAGTGAAGAATGGAGTGTG (reverse complemental)	Staphylococcus epidermidis
				Tuf 7	23 54	ATATGACTTCCCAGGTGAC GTCACCTGGGAAGTCATAT (reverse complemental)	General T uf gene
Tuf 8	24 55	GTAGATACTTACATTCCA TGGAATGTAAGTATCTAC (reverse complemental)	Streptococcus aureus
				Tuf 9	25 56	GTTGATGATTACATTCCA TGGAATGTAATCATCAAC (reverse complemental)	Epidermis grape ball mattress
Tuf 10	26 57	CCATTCTTCACTAACTACCGC GCGGTAGTTAGTGAAGAATGG (reverse complemental)	Staphylococcus saprophyticus imitation staphylococcus (S. simulans)
				Tuf 11	27 58	CATACGCCATTCTTCACTAACT AGTTAGTGAAGAATGGCGTATG (reverse complemental)	Staphylococcus saprophyticus
Tuf 15	28 59	CCATTCTTCTCTAACTATCGT ACGATAGTTAGAGAAGAATGG (reverse complemental)	Human-like staphylococcus
				Tuf 16	29 60	CCATTCTTCACAAACTATCGT ACGATAGTTTGTGAAGAATGG (reverse complemental)	Staphylococcus haemolyticus
Tuf 17	30 6l	CCATTCTTCAGTAACTATCGC GCGATAGTTACTGAAGAATGG (reverse complemental)	Staphylococcus cohnis (S. cohnii)
				Tuf 18	31 62	CCATTCTTCAGTAACTACCGC GCGGTAGTTACTGAAGAATGG (reverse complemental)	S.warneri head staphylococcus (S. capitis)
Tuf 19	32 63	CCATTCTTCTCAAACTACCGC GCGGTAGTTTGAGAAGAATGG (reverse complemental)	Lyons staphylococcus (S. lugdunensis)
				Tuf 20	33 64	CCATTCTTCTCTAACTACCGT ACGGTAGTTAGAGAAGAATGG (reverse complemental)	Staphylococcus auricularis (S. auricularis)

Tuf 21	34 65	CATACGCCATTCTTCAGTAACT AGTTACTGAAGAATGGCGTATG (reverse complemental)	Staphylococcus cohnis
				Tuf 22	35 66	CACACTCCATTCTTCAGTAACT AGTTACTGAAGAATGGAGTGTG (reverse complemental)	S.warneri head staphylococcus
Tuf 23	36 67	CATACTCCATTCTTCACTAACT AGTTAGTGAAGAATGGAGTATG (reverse complemental)	The imitation staphylococcus
				Tuf 24	37 68	CATACACCATTCTTCTCAAACT AGTTTGAGAAGAATGGTGTATG (reverse complemental)	Lyons staphylococcus
Tuf 25	38 69	CATACTCCATTCTTCTCTAACT AGTTAGAGAAGAATGGAGTATG (reverse complemental)	Human-like staphylococcus
				Tuf 26	39 70	CACACTCCATTCTTCACAAACT AGTTTGTGAAGAATGGAGTGTG (reverse complemental)	Staphylococcus haemolyticus
Tuf 27	40 71	CACACTCCATTCTTCTCTAACT AGTTAGAGAAGAATGGAGTGTG (reverse complemental)	Staphylococcus auricularis
				MecA 1	41 72	TCGATGGTAAAGGTTGGC GCCAACCTTTACCATCGA (reverse complemental)	The mecA gene
MecA 2	42 73	ATGGCATGAGTAACGAAGAATATA TATTGTATTCGTTACTCATGCCAT (reverse complemental)	The mecA gene
				MecA 3	43 74	AAAGAACCTCTGCTCAACAAG CTTGTTGAGCAGAGGTTCTTT (reverse complemental)	The mecA gene
MecA 4	44 75	GCACTTGTAAGCACACCTTCAT ATGAAGGTGTGCTTACAAGTGC (reverse complemental)	The mecA gene
				MecA 6	45 76	TTCCAGATTACAACTTCACCA TGGTGAAGTTGTAATCTGGAA (reverse complemental)
16S 12	46 77	GTTCCTCCATATCTCTGCG CGCAGAGATATGGAGGAAC (reverse complemental)	16s rRNA
				16S 13	47 78	ATTTCACCGCTACACATG CATGTGTAGCGGTGAAAT (antisense is to mending)	16s rRNA

Embodiment 4

With the mec A gene order of golden nanometer particle probe in detecting from bacterial genomes DNA

In the present embodiment, described in array layout, used method based on the detection mec A gene order of golden nanometer particle detection.Have as the mocroarray plate of the mecA 2 of capture probe and mecA 6 oligonucleotide golden nanometer particle and use with mecA 4 oligonucleotide exploratory probe marks.Mocroarray plate, capture probe and exploratory probe are according to embodiment 3 described method preparations.

Be used to show existence with the mec A DNA of the transparency carrier hybridization of three composition sandwich test forms as the golden nanometer particle that is attached with oligonucleotide probe (diameter 13nm) of method preparation as described in the embodiment 3.Then, the nanoparticle that is attached with probe oligonucleotides with separate genomic dna target thing cohybridization from X-1497 resistance (MecA+) or methicillin-sensitivity (MecA-) streptococcus aureus bacterial cell on these substrates.Therefore, nanoparticle existence demonstration has from the teeth outwards detected mec A gene order, sees Figure 16.At the target thing amount of being tested (250ng (7.5E7 copy)-1 μ g (3.0E8)), can not observe the nanoparticle that adheres to bore hole.For the ease of observing to hybridizing in the nanoparticle of substrate surface, adopted method for amplifying signal, use Resorcinol catalytic reduction silver ions in the method, on the slide glass surface, form silver metal.Although this method has been used to amplify protein and antibody conjugated golden nanometer particle (Hacker in histological chemistry's microscopy research, in Colloidal Gold:Principles, Methods, and Applications, M.A.Hayat, Ed. (Academic Press, SanDiego, 1989)), vol.1, chap.10; Zehbe etc., Am.J.Pathol.150,1553 (1997)), but its purposes in quantitatively DNA hybridization is tested is new (Tomlinson etc., Anal.Biochem., 171:217 (1988)).This method not only makes the low-down nanoparticle probe of surface coverage observe by flat bed scanner or with bore hole, also allows quantitatively dyeing district to go up the target thing hybridization of dying the Au probe scattered light of amplification based on silver.Clearly, on the amount of each genomic dna of test, to obtain strength of signal much higher from containing methicillin-sensitivity staphylococcus aureus gene group DNA for the signal intensity ratio that obtains from the sample that contains X-1497 resistance staphylococcus aureus gene group DNA.This shows that this detection side's science of law can be used for the specific detection of mec A gene in the presence of complicated bacterial genomes DNA background, see Figure 16.This result is the unusual characteristics of nanoparticle oligonucleotide conjugate thing, and it makes the hypersensitive of nucleic acid and selectivity detection become possibility.Should be noted that also this method does not need zymetology target thing or method for amplifying signal, it provides the novel method that detects gene from bacterial genomes DNA sample.

(a) target dna preparation

Separation from the total genomic dna of the purifying of staphylococcus bacterial cell available from ATCC (AmericanType Culture Collection).Described in embodiment 5 (face as follows), before hybridization array, total genomic dna passes through the ultrasonic degradation fragmentation to shear dna molecular.

(b) MecA gene test test

(ii) testing method

Content range is reaction mixture preparation in 1 * hybridization buffer (5 * SSC, 0.05%Tween 20) of bacterial genomes DNA and the lnM nanoparticle probe of 250ng-1 μ g.With reaction mixture be heated to 95 ℃ 5 minutes.Subsequently, the reaction mixture of 10-25 μ L is added to microarray surface, hybridization is 2 hours under 40 ℃ and 90% the relative humidity condition.Microarray surface was at room temperature washed 30 seconds with 5 * SSC, 0.05%Tween 20.Dry microarray, and with business level silver toughener solution (silverenhancer solution) (Sigma St.Louis) carries out silver and contacts and developed the color 4 minutes for silver-colored toughener test kit (Silver Enhancer Kit), Catalog No.SE-100.The microarray dish washing of then, silver being dyed, dry also with Arrayworx  scanner (Model No.AWE, Applied Precision, Inc., Issaquah, WA) imaging.

Embodiment 5

Tuf probe with bacterial genomes DNA and golden nanometer particle mark carries out staphylococcus species formation analysis

In the present embodiment, by distinguishing that carrying out the staphylococcus species with streptococcus aureus and the corresponding Tuf gene order of staphylococcus epidermidis bacterial classification forms analysis.Come the sequence-specific of array of display as positive control from Tuf 372bp pcr amplification of the total genomic dna amplification of streptococcus aureus and staphylococcus epidermidis bacterial cell from separation.In independent hybridization,, and itself and mocroarray plate hybridized the total genomic dna fragmentation that separates from streptococcus aureus and staphylococcus epidermidis bacterial cell.Mocroarray plate comprises combination Tuf 3, Tuf 4 or Tuf 5 and Tuf 6 capture probes thereon.The golden nanometer particle of Tuf 2 oligonucleotide marks is as exploratory probe.The method of describing according to embodiment 3 prepares mocroarray plate, catches and exploratory probe.Prepare Tuf 372bp amplicon with conventional P CR amplification method.

(a) target dna preparation

Be prepared as follows genomic dna: the genomic dna that separates the staphylococcus bacterial cell of cultivating certainly is available from ATCC (American Type Culture Collection).Is hydration once more in the water of no DNA enzyme of 200 μ l with the dry DNA of＞10 μ g part in volume.Use Misonix then, ultrasonic cell disintegration instrument (Ultrasonic cell disruptor) XL Farmingdale, NY, with 12 times, the pulse of 2 watts of each about 0.5 second power is with its ultrasonic degradation.Use the total DNA concentration of commercially available Picogreen kit measurement from Molecular Probes, and on Tecanspectrafluor plus fluorescent plate reader reading.In the enterprising line scanning analysis of Agilent 2100 biological analysers (Bioanalyzer) (smear analysis), the size of the dna fragmentation that records thus is average 1.5Kb.With conventional P CR amplification technique, positive control tuf gene 372 base pair pcr amplification from streptococcus aureus or staphylococcus epidermidis gene group DNA, have been prepared.

(b) Tuf gene test testing method

In independent hybridization hole, to separate from staphylococcus epidermidis or streptococcus aureus bacterial cell (8.0E07 copy,～250ng) fragmentation total genomic dna and 1nM nanoparticle probe is mixed in the 1x hybridization buffer (5X SSC, 0.05%Tween 20).The pcr amplification Tuf gene fragment of same genome DNA sample is as positive control, is mixed in the independent hybridization hole on the glass slide with probe and damping fluid.With reaction mixture be heated to 95 ℃ 5 minutes.Subsequently, 50 μ l reaction mixtures are added to microarray surface, hybridization is 1.5 hours under 45 ℃ and the 90% relative humidity condition.Under the microarray surface room temperature at 0.5M NaNO ₃Middle flushing 30 seconds.Dry microarray, and with business level silver toughener solution (Silver Enhancer Kit, Catalog No.SE-100, Sigma, St., Louis MO) carries out silver and contacts and developed the color 4 minutes.Wash the mocroarray plate that silver dyes then, drying, (Issaquah WA) dyes the silver on the array scattered light imaging and the quantification of amplifying the nanoparticle probe for Model No.AWE, Applied Precision with Arrayworx  scanner.

The result of Tuf3 and Tuf4 capture probe is as Figure 17 (a) with (b), and the result of Tuf5 and Tuf6 capture probe is as Figure 17 (c) with (d).Use Tuf3 and Tuf4 capture probe group, when genomic dna and hybridization array, observed on the array specific signals corresponding to staphylococcus kind streptococcus aureus and staphylococcus epidermidis.This has convincingly demonstrated, and in the presence of total genomic dna, distinguishes reduced complexity and amplification that these sequences that are closely related do not need to be undertaken by PCR tuf gene target thing.Use Tuf3 and Tuf4 capture probe group, also observed signal, but some cross reactions have been arranged, cause lower recognition rate thus with the mispairing acquisition sequence corresponding to suitable species.This shows that sequences Design is vital for the accurate evaluation of species.

Embodiment 6

Carrying out the staphylococcus species with pcr amplification and golden nanometer particle mark mec A and Tuf oligonucleotide as exploratory probe forms and analyzes and the X-1497 resistance is tested

In the present embodiment, designed being used to identifies that the array of staphylococcic genus, kind and antibiotics resistance situation is by making from the sequence of 16S rRNA gene (genus), Tuf gene (specific specificity of streptococcus aureus, staphylococcus epidermidis and Staphylococcus saprophyticus is caught kind) and mec A gene (antibiotics resistance situation).Notice that staphylococcus epidermidis and Staphylococcus saprophyticus capture probe have only a nucleotide difference, and streptococcus aureus and staphylococcus epidermidis capture probe there is the difference of three Nucleotide.Mocroarray plate comprises all following sequence: 16S 12, mecA 6, Tuf 3, Tuf 4, Tuf 10 capture probes and a combination negative hybrid capture probe thereon.The Tuf 2 of golden nanometer particle mark, mecA 4 and 16S 12 probes are as exploratory probe.Mocroarray plate, capture probe and exploratory probe are according to embodiment 4 described method preparations.Specificity with pcr amplification from the gene order hot-wire array of various X-1497s resistance and methicillin-sensitivity staphylococcus species (streptococcus aureus, staphylococcus epidermidis, Staphylococcus saprophyticus).The specific PCR amplification gene fragment that is used to test is (mecA 281,16S 451 and Tuf 372) as shown in figure 18.The Tuf gene order that comes from different staphylococcus species shown in Figure 180 obtains from GenBank.

The preparation of target thing:

Use Standard PC R amplification method to prepare the gene product of pcr amplification.

Test:

Each reaction system comprises every kind of nanoparticle probe of 50 μ l 5x SSC, 0.05%Tween 20,0.01%BSA, 200pM and every kind of target amplification of 15% methane amide and 750pM.Reagent hybridization 1 hour under 40 ℃ and 90% humidity condition.Under the room temperature at 0.5M NaNO ₃Middle washing microarray surface 30 seconds.Dry microarray, and with business level silver toughener solution (Silver Enhancer Kit, Catalog No.SE-100, Sigma, St.Louis MO) carries out silver and contacts and developed the color 4 minutes.Wash the mocroarray plate that silver dyes then, drying is with Arrayworx  scanner (Model No.AWE, AppliedPrecision, Issaquah, WA) imaging.

The result is as Figure 19 (a) with (b).When adopting Standard PC R amplification method, correctly identified the species and the X-1497 resistance situation of selected five staphylococcus samples (opinion) with sequence-specific pcr amplification of array of display as following table 3.

Table 3

ATCC sample ID#	Describe
		35556 700699 12228 35984 15305	The streptococcus aureus streptococcus aureus, Mu50-X-1497 resistance staphylococcus epidermidis staphylococcus epidermidis, many antibiotics resistances of RP62A-Staphylococcus saprophyticus

Embodiment 7

Use mecA, 16S and the Tuf probe of genomic dna and golden nanometer particle mark to carry out staphylococcus species formation analysis and the test of X-1497 resistance

Among this embodiment, use separation to carry out the evaluation of staphylococcic genus, kind and antibiotics resistance situation from the total genomic dna of the bacterial cell of streptococcus aureus and staphylococcus epidermidis.Described in the feature such as above-mentioned table 3 that the genome DNA sample of being tested is represented by ATCC.The mocroarray plate and the exploratory probe of test usefulness also are used for present embodiment among the embodiment 6.Prepare mocroarray plate and catch and exploratory probe with the method for describing among the embodiment 3.Method according to embodiment 5 prepares genome DNA sample.Each reaction system comprises the every kind of nanoparticle probe of 50 μ l 5x SSC, 0.05%Tween 20,0.01%BSA, 200pM and the ultrasonic degradation genomic dna of 15% methane amide and 3.3ng/ μ l.Reagent hybridization 2 hours under 40 ℃ and 90% humidity condition.Under the room temperature at 0.5M NaNO ₃Middle washing microarray surface 30 seconds.Dry microarray, and with business level silver toughener solution (Silver EnhancerKit, Catalog No.SE-100, Sigma, St. Louis) carries out silver and contacts and developed the color 4 minutes.Wash the mocroarray plate that silver dyes then, drying is with Arrayworxt  scanner (Model No.AWE, AppliedPrecision, Issaquah, WA) imaging.

The result as shown in figure 20.Clearly, only to exceed the net signal intensity of three times of standard deviations of background in each sample, correctly identified the staphylococcus bacterial classification and the antibiotics resistance situation of three genomic dnas being tested in correct capture probe site.This tests demonstration, when aureus gene group DNA and hybridization array, and by silver when dying the golden nanometer particle probe mark of amplification, even if the sudden change of single Nucleotide also can be detected in the Tuf gene.Therefore, need not new detection side's science of law of any target amplification (as PCR) or signal amplification (horseradish peroxidase) method by this, can form analysis having only few biology microorganism carry out species in the given gene order to single nucleotide difference based on enzyme.

The separation of known quantity is dropped in the test system (assay) from the total genomic dna of X-1497 resistance aureus cell, measure net signal intensity,, measure the test susceptibility with this as Figure 21 from mecA gene trap probe.But minimum detection limit is 34ng, corresponding to about 1,000 ten thousand genomes copy.The further optimization of described detection method should detect the genomic dna of less amount.

Be to be understood that aforementioned disclosed content focuses on some specific embodiments of the present invention, all modifications of equal value with it or replacement all comprise within the spirit and scope of the present invention, as listed in the additional claim.

Claims (167)

1. the method for target nucleic acid sequence in the test sample, described sample comprises the nucleic acid molecule with respect to the higher biological complex degree of nucleic acid molecule of amplification, the target nucleic acid sequence has at least a Nucleotide different with one or more nucleotide sequences, and the method comprising the steps of:

A) provide the addressable substrate that is combined with capture oligo, wherein capture oligo has at least a portion complementary sequence with the first part of target nucleic acid sequence;

B) provide and comprise the exploratory probe of surveying oligonucleotide, wherein survey oligonucleotide and have at least a portion complementary sequence with the second section of the target nucleic acid sequence of step (a);

C) can effectively hybridize with the first part of target nucleic acid sequence at capture oligo, exploratory probe can effectively be hybridized with the second section of target nucleic acid sequence, and allow to distinguish under the condition of target sequence described one or more nucleotide sequences different with having at least one Nucleotide, sample is contacted with exploratory probe with substrate; With

Whether d) detect capture oligo and exploratory probe hybridizes with first and second parts of target nucleic acid sequence.

2. the process of claim 1 wherein that the target nucleic acid sequence comprises single nucleotide polymorphism.

3. the process of claim 1 wherein that the difference of single Nucleotide is incorporated into the capture oligo identification of substrate.

4. the process of claim 1 wherein that the difference of single Nucleotide is detected oligonucleotide identification.

5. the process of claim 1 wherein that target nucleic acid molecules comprises the RNA of genomic dna, geneome RNA, expression, plasmid DNA, plastosome or other cell organelles DNA, free cell DNA, viral DNA or viral RNA or above-mentioned two or more mixture.

6. the process of claim 1 wherein that substrate comprises many capture oligos, wherein each can both discern different single nucleotide polymorphism.

7. the process of claim 1 wherein that sample comprises more than one nucleic acid target material, wherein each nucleic acid target material all comprises one or more different single nucleotide polymorphism.

8. the process of claim 1 wherein provides the exploratory probe of one or more types, and wherein each exploratory probe all has bonded detection oligonucleotide with it, can hybridize with different nucleic acid target material.

9. the method for claim 1, wherein sample is contacted with exploratory probe, so that be present in nucleic acid target material in the sample and the detection oligonucleotide hybridization on the exploratory probe, the nucleic acid target material that will be incorporated into exploratory probe then with substrate contacts so that the hybridization of the capture oligo on nucleic acid target material and the substrate.

10. the method for claim 1, wherein sample and substrate contacts so that the nucleic acid target material and the capture oligo that are present in the sample are hybridized, the nucleic acid target material that will be incorporated into capture oligo then contacts with exploratory probe, so that the detection oligonucleotide hybridization on nucleic acid target material and the exploratory probe.

11. the process of claim 1 wherein that sample simultaneously and exploratory probe and substrate contacts.

12. the process of claim 1 wherein that surveying oligonucleotide comprises detectable marker.

13. the method for claim 12, wherein snoop tag allows to survey by photonics, electronics, acoustics, optoacoustic, gravity, electrochemistry, electric light (electro-optic), mass spectrum, zymetology, chemistry, biological chemistry or physics means.

14. the method for claim 12, wherein mark is a fluorescence.

15. the method for claim 12, wherein mark is luminous.

16. the method for claim 12, wherein mark is phosphorescent.

17. the method for claim 12, wherein mark is radioactive.

18. the method for claim 12, wherein mark is a nanoparticle.

19. the method for claim 12, wherein mark is a dendrimer.

20. the method for claim 12, wherein mark is a molecule aggregates.

21. the method for claim 12, wherein mark is a quantum dot.

22. the method for claim 12, wherein mark is a pearl.

23. the process of claim 1 wherein that exploratory probe is to be combined with the nanoparticle probe of surveying oligonucleotide.

24. the method for claim 23, wherein nanoparticle is made by noble metal.

25. the method for claim 24, wherein nanoparticle is made by gold or silver.

26. the method for claim 25, wherein nanoparticle is made of gold.

27. the method for claim 23, wherein detection comprises that substrate and silver are dyed the district to be contacted.

28. the method for claim 23, wherein detection comprises the light of detection by the nanoparticle scattering.

29. the method for claim 23 wherein detects and comprises with photoscanner and observing.

30. the method for claim 23 wherein detects and comprises with flat bed scanner and observing.

31. the method for claim 29 or 30, wherein scanner is connected on the computer, is mounted with the software that can calculate gray-scale value on the computer, and the gray-scale value that is calculated provides the quantitative values of the amount of the nucleic acid that detects.

32. the method for claim 23 wherein is attached to oligonucleotide on the substrate between two electrodes, nanoparticle is made by electrical conductor material, and step (d) comprises the change that detects electroconductibility.

33. the method for claim 32, wherein electrode is made of gold, and nanoparticle also is made of gold.

34. the method for claim 32, wherein substrate dyes to distinguish with silver and contacts, and has produced the change of electroconductibility.

35. the method for claim 23, wherein each the many oligonucleotide that can both discern different target nucleic acid sequences is attached in the array of spots on the substrate, each oligonucleotide spot is all between two electrodes, nanoparticle is made by electrical conductor material, and step (d) comprises the change that detects electroconductibility.

36. the method for claim 35, wherein electrode is made of gold, and nanoparticle also is made of gold.

37. the method for claim 35, wherein substrate dyes to distinguish with silver and contacts to produce the change of electroconductibility.

38. the method for one or more target nucleic acid sequences in the test sample, described sample comprises the nucleic acid molecule than the nucleic acid molecule biological complex Du Genggao of amplification, in one or more target nucleic acid sequences each all has at least a Nucleotide different with known nucleic acid sequence, and the method comprising the steps of:

A) provide the addressable substrate that is combined with many capture oligos, wherein capture oligo has one or more part complementary sequences with one or more target nucleic acid sequences;

B) provide one or more to comprise the exploratory probe of surveying oligonucleotide, wherein survey oligonucleotide and have one or more part complementary sequences with one or more target nucleic acid sequences of step (a);

C) can effectively hybridize with one or more parts of one or more target nucleic acid sequences at capture oligo, exploratory probe can effectively be hybridized with the part of one or more target nucleic acid sequences, and allow to distinguish under the condition of the target nucleic acid sequence with at least one nucleotide difference, sample is contacted with exploratory probe with substrate; With

D) detect any capture oligo and exploratory probe whether with any target nucleic acid sequence hybridization.

39. the method for claim 38, wherein the target nucleic acid sequence comprises single nucleotide polymorphism.

40. the method for claim 38, wherein the difference of single Nucleotide is discerned by the capture oligo that is incorporated into substrate.

41. the method for claim 38, wherein the difference of single Nucleotide is discerned by surveying oligonucleotide.

42. the method for claim 38, wherein target nucleic acid molecules comprises RNA, plasmid DNA, plastosome or other cell organelles DNA, free cell DNA, viral DNA or viral RNA or above-mentioned two or more mixture of genomic dna, geneome RNA, expression.

43. the method for claim 38, wherein substrate comprises many capture oligos, and wherein each capture oligo can both be discerned different single nucleotide polymorphism.

44. the method for claim 38, wherein sample comprises more than one nucleic acid target material, and wherein each all comprises different single nucleotide polymorphism.

45. the method for claim 38 wherein provides the exploratory probe of one or more types, wherein each nucleic acid target material all is combined with the detection oligonucleotide, can with different nucleic acid target material hybridization.

46. the method for claim 38, wherein sample is contacted with exploratory probe, so that be present in nucleic acid target material in the sample and the detection oligonucleotide hybridization on the exploratory probe, the nucleic acid target material that will be incorporated into exploratory probe then with substrate contacts so that the hybridization of the capture oligo on nucleic acid target material and the substrate.

47. the method for claim 38, wherein sample and substrate contacts so that the nucleic acid target material and the capture oligo that are present in the sample are hybridized, the nucleic acid target material that will be incorporated into capture oligo then contacts with exploratory probe, so that the detection oligonucleotide hybridization on nucleic acid target material and the exploratory probe.

48. the method for claim 38, wherein sample while and exploratory probe and substrate contacts.

49. the method for claim 38, wherein exploratory probe comprises detectable marker.

50. the method for claim 49, wherein snoop tag allows to survey by photonics, electronics, acoustics, optoacoustic, gravity, electrochemistry, electric light, mass spectrum, zymetology, chemistry, biological chemistry or physics means.

51. the method for claim 49, wherein mark is a fluorescence.

52. the method for claim 49, wherein mark is luminous.

53. the method for claim 49, wherein mark is phosphorescent.

54. the method for claim 49, wherein mark is radioactive.

55. the method for claim 49, wherein mark is a nanoparticle.

56. the method for claim 49, wherein mark is a dendrimer.

57. the method for claim 49, wherein mark is a molecule aggregates.

58. the method for claim 49, wherein mark is a quantum dot.

59. the method for claim 49, wherein mark is a pearl.

60. the method for claim 38, wherein exploratory probe is to be combined with the nanoparticle probe of surveying oligonucleotide.

61. the method for claim 60, wherein nanoparticle is made by noble metal.

62. the method for claim 61, wherein nanoparticle is made by gold or silver.

63. the method for claim 62, wherein nanoparticle is made of gold.

64. the method for claim 60, wherein detection comprises that substrate and silver are dyed the district to be contacted.

65. the method for claim 60, wherein detection comprises the light of observation by the nanoparticle scattering.

66. the method for claim 60 wherein detects and comprises with photoscanner and observing.

67. the method for claim 60 wherein detects and comprises with flat bed scanner and observing.

68. the method for claim 66 or 67, wherein scanner is connected on the computer, is mounted with the software that can calculate gray-scale value on the computer, and the gray-scale value that is calculated provides the quantitative values of the amount of the nucleic acid that detects.

69. the method for claim 60 wherein is attached to oligonucleotide on the substrate between two electrodes, nanoparticle is made by electrical conductor material, and step (d) comprises the change that detects electroconductibility.

70. the method for claim 69, wherein electrode is made of gold, and nanoparticle also is made of gold.

71. the method for claim 69, wherein substrate dyes to distinguish with silver and contacts to produce the change of electroconductibility.

72. the method for claim 60, wherein each the many oligonucleotide that can both discern different target nucleic acid sequences is attached in the array of spots on the substrate, each oligonucleotide spot is all between two electrodes, nanoparticle is made by electrical conductor material, and step (d) comprises the change that detects electroconductibility.

73. the method for claim 72, wherein electrode is made of gold, and nanoparticle also is made of gold.

74. the method for claim 72, wherein substrate dyes to distinguish with silver and contacts to produce the change of electroconductibility.

75. the method for single nucleotide polymorphism in the evaluation sample, described sample comprise the nucleic acid molecule than the nucleic acid molecule biological complex Du Genggao of amplification, the method comprising the steps of:

A) provide the addressable substrate that is combined with at least one capture oligo, wherein said at least one capture oligo has and at least a portion complementary sequence that comprises the nucleic acid target material of specific polymorphism;

B) provide and be combined with the exploratory probe of surveying oligonucleotide, wherein survey oligonucleotide and have at least a portion complementary sequence with the nucleic acid target material of step (a);

C) can effectively hybridize with nucleic acid target material at capture oligo, exploratory probe can effectively be hybridized with nucleic acid target material, and allows to distinguish under the condition of the target thing with single nucleotide difference, and sample is contacted with exploratory probe with substrate; With

Whether d) detect capture oligo and exploratory probe hybridizes with nucleic acid target material.

76. the method for claim 75, wherein polymorphism is discerned by the capture oligo that is incorporated into substrate.

77. the method for claim 75, wherein polymorphism is discerned by surveying oligonucleotide.

78. the method for claim 75, wherein the nucleic acid molecule in the sample comprises RNA, plasmid DNA, plastosome or other cell organelles DNA, free cell DNA, viral DNA or viral RNA or above-mentioned two or more mixture of genomic dna, geneome RNA, expression.

79. the method for claim 75, wherein substrate comprises many capture oligos, and wherein each capture oligo can both be discerned different single nucleotide polymorphism.

80. the method for claim 75, wherein sample comprises more than one nucleic acid target material, and wherein each nucleic acid target material all comprises one or more different single nucleotide polymorphism.

81. the method for claim 75 wherein provides the exploratory probe of one or more types, wherein each exploratory probe all is combined with the detection oligonucleotide, can with different nucleic acid target material hybridization.

82. the method for claim 75, wherein sample is contacted with exploratory probe, so that be present in nucleic acid target material in the sample and the detection oligonucleotide hybridization on the exploratory probe, the nucleic acid target material that will be incorporated into exploratory probe then with substrate contacts so that the hybridization of the capture oligo on nucleic acid target material and the substrate.

83. the method for claim 75, wherein sample and substrate contacts so that the nucleic acid target material and the capture oligo that are present in the sample are hybridized, the nucleic acid target material that will be incorporated into capture oligo then contacts with exploratory probe, so that the detection oligonucleotide hybridization on nucleic acid target material and the exploratory probe.

84. the method for claim 75, wherein sample while and exploratory probe and substrate contacts.

85. the method for claim 75, wherein exploratory probe comprises detectable marker.

86. the method for claim 85, wherein snoop tag allows to survey by photonics, electronics, acoustics, optoacoustic, gravity, electrochemistry, electric light, mass spectrum, zymetology, chemistry, biological chemistry or physics means.

87. the method for claim 85, wherein mark is a fluorescence.

88. the method for claim 85, wherein mark is a nanoparticle.

89. the method for claim 85, wherein mark is luminous.

90. the method for claim 85, wherein mark is phosphorescent.

91. the method for claim 85, wherein mark is radioactive.

92. the method for claim 85, wherein mark is a dendrimer.

93. the method for claim 85, wherein mark is a molecule aggregates.

94. the method for claim 85, wherein mark is a quantum dot.

95. the method for claim 85, wherein mark is a pearl.

96. the method for claim 75, wherein exploratory probe is to be combined with the nanoparticle probe of surveying oligonucleotide.

97. the method for claim 96, wherein nanoparticle is made by noble metal.

98. the method for claim 97, wherein nanoparticle is made by gold or silver.

99. the method for claim 98, wherein nanoparticle is made of gold.

100. the method for claim 96, wherein detection comprises that substrate and silver are dyed the district to be contacted.

101. the method for claim 96, wherein detection comprises the light of detection by the nanoparticle scattering.

102. the method for claim 96 wherein detects and comprises with photoscanner and observing.

103. the method for claim 96 wherein detects and comprises with flat bed scanner and observing.

104. the method for claim 102 or 103, wherein scanner is connected in computer, is mounted with the software that can calculate gray-scale value on the computer, and the gray-scale value that is calculated provides the quantitative values of the amount of the nucleic acid that detects.

105. the method for claim 96 wherein is attached to oligonucleotide on the substrate between two electrodes, nanoparticle is made by electrical conductor material, and step (d) comprises the change that detects electroconductibility.

106. the method for claim 105, wherein electrode is made of gold, and nanoparticle also is made of gold.

107. the method for claim 105, wherein substrate dyes to distinguish with silver and contacts to produce the change of electroconductibility.

108. the method for claim 96, wherein each the many oligonucleotide that can both discern different single nucleotide polymorphism is attached in the array of spots on the substrate, each oligonucleotide spot is all between two electrodes, nanoparticle is made by electrical conductor material, and step (d) comprises the change that detects electroconductibility.

109. the method for claim 108, wherein electrode is made of gold, and nanoparticle also is made of gold.

110. the method for claim 109, wherein substrate dyes to distinguish with silver and contacts to produce the change of electroconductibility.

111. the method for one or more single nucleotide polymorphism in the evaluation sample, described sample comprise the nucleic acid molecule than the nucleic acid molecule biological complex Du Genggao of amplification, the method comprising the steps of:

A) provide the addressable substrate that is combined with many capture oligos, wherein capture oligo has a plurality of part complementary sequences with nucleic acid target material, and each described part all comprises specific polymorphism;

B) provide and comprise one or more exploratory probes of surveying oligonucleotide, wherein survey oligonucleotide and have and one of them at least a portion complementary sequence of the nucleic acid target material of step (a), this nucleic acid target material is not discerned by the capture oligo on the substrate;

C) can effectively hybridize with a plurality of parts of nucleic acid target material at capture oligo, exploratory probe can effectively be hybridized with nucleic acid target material, and allows to distinguish under the condition of the target thing with single nucleotide difference, and sample is contacted with exploratory probe with substrate; With

Whether d) detect any capture oligo and exploratory probe hybridizes with any nucleic acid target material.

112. the method for claim 111, wherein polymorphism is discerned by the capture oligo that is incorporated into substrate.

113. the method for claim 111, wherein polymorphism is discerned by surveying oligonucleotide.

114. the method for claim 111, wherein the nucleic acid molecule in the sample comprises RNA, plasmid DNA, plastosome or other cell organelles DNA, free cell DNA, viral DNA or viral RNA or above-mentioned two or more mixture of genomic dna, geneome RNA, expression.

115. the method for claim 111, wherein substrate comprises many capture oligos, and wherein each can both discern one or more different single nucleotide polymorphism.

116. the method for claim 111, wherein sample comprises more than one nucleic acid target material, and wherein each all comprises different single nucleotide polymorphism.

117. the method for claim 111 wherein provides the exploratory probe of one or more types, wherein each all is combined with the detection oligonucleotide, can with different nucleic acid target material hybridization.

118. the method for claim 111, wherein sample is contacted with exploratory probe, so that be present in nucleic acid target material in the sample and the detection oligonucleotide hybridization on the exploratory probe, the nucleic acid target material that will be incorporated into exploratory probe then with substrate contacts so that the hybridization of the capture oligo on nucleic acid target material and the substrate.

119. the method for claim 111, wherein sample and substrate contacts so that the nucleic acid target material and the capture oligo that are present in the sample are hybridized, the nucleic acid target material that will be incorporated into capture oligo then contacts with exploratory probe, so that the detection oligonucleotide hybridization on nucleic acid target material and the exploratory probe.

120. the method for claim 111, wherein sample while and exploratory probe and substrate contacts.

121. the method for claim 111 is wherein surveyed oligonucleotide and is comprised detectable marker.

122. the method for claim 121, wherein snoop tag allows to survey by photonics, electronics, acoustics, optoacoustic, gravity, electrochemistry, electric light, mass spectrum, zymetology, chemistry, biological chemistry or physics means.

123. the method for claim 121, wherein mark is a fluorescence.

124. the method for claim 121, wherein mark is luminous.

125. the method for claim 121, wherein mark is phosphorescent.

126. the method for claim 121, wherein mark is radioactive.

127. the method for claim 121, wherein mark is a nanoparticle.

128. the method for claim 121, wherein mark is a dendrimer.

129. the method for claim 121, wherein mark is a molecule aggregates.

130. the method for claim 121, wherein mark is a quantum dot.

131. the method for claim 121, wherein mark is a pearl.

132. the method for claim 111, wherein exploratory probe is to be combined with the nanoparticle probe of surveying oligonucleotide.

133. the method for claim 132, wherein nanoparticle is made by noble metal.

134. the method for claim 133, wherein nanoparticle is made by gold or silver.

135. the method for claim 134, wherein nanoparticle is made of gold.

136. the method for claim 132, wherein detection comprises that substrate and silver are dyed the district to be contacted.

137. the method for claim 132, wherein detection comprises the light of detection by the nanoparticle scattering.

138. the method for claim 132 wherein detects and comprises with photoscanner and observing.

139. the method for claim 132 wherein detects and comprises with flat bed scanner and observing.

140. the method for claim 138 or 139, wherein scanner is connected in computer, is mounted with the software that can calculate gray-scale value on the computer, and the gray-scale value that is calculated provides the quantitative values of the amount of the nucleic acid that detects.

141. the method for claim 132 wherein is attached to oligonucleotide on the substrate between two electrodes, nanoparticle is made by electrical conductor material, and step (d) comprises the change that detects electroconductibility.

142. the method for claim 141, wherein electrode is made of gold, and nanoparticle also is made of gold.

143. the method for claim 141, wherein substrate dyes to distinguish with silver and contacts to produce the change of electroconductibility.

144. the method for claim 141, wherein each the many oligonucleotide that can both discern different single nucleotide polymorphism is attached in the array of spots on the substrate, each oligonucleotide spot is all between two electrodes, nanoparticle is made by electrical conductor material, and step (d) comprises the change that detects electroconductibility.

145. the method for claim 144, wherein electrode is made of gold, and nanoparticle also is made of gold.

146. the method for claim 146, wherein substrate dyes to distinguish with silver and contacts to produce the change of electroconductibility.

147. claim 1,38,75 or 111 method, wherein higher biological complex degree is greater than about 50,000.

148. claim 1,38,75 or 111 method, wherein higher biological complex degree are about 50,000-50,000,000,000.

149. claim 1,38,75 or 111 method, wherein higher biological complex degree is about 1,000,000,000.

150. the method for claim 1 or 38, wherein the target nucleic acid sequence is the part of biology microbial gene.

151. the method for claim 1 or 38, wherein the target nucleic acid sequence is the part of aureus gene.

152. the method for claim 151, wherein staphylococcus is streptococcus aureus, staphylococcus haemolyticus, staphylococcus epidermidis, Lyons staphylococcus, human-like staphylococcus or Staphylococcus saprophyticus.

153. the method for claim 151, wherein the target nucleic acid sequence is the part of Tuf gene.

154. the method for claim 151, wherein the target nucleic acid sequence is the part of femA gene.

155. the method for claim 151, wherein the target nucleic acid sequence is the part of 16S rRNA gene.

156. the method for claim 151, wherein the target nucleic acid sequence is the part of hsp60 gene.

157. the method for claim 151, wherein the target nucleic acid sequence is the part of sodA gene.

158. the method for claim 1 or 38, wherein the target nucleic acid sequence is the part of mecA gene.

159. the method for 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 IDNO: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, SEQID 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 IDNO: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 IDNO: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 IDNO: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 listed sequence among the SEQ IDNO:78.

160. the method for claim 1 or 38, wherein, one of them is surveyed oligonucleotide and 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 IDNO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQID 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 IDNO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQID 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 IDNO: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 IDNO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, or listed sequence among the SEQ ID NO:78.

161. the process of claim 1 wherein that capture oligo comprises SEQ ID NO:17, SEQID 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, SEQID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ IDNO:32, SEQ ID NO:33, SEQID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQID NO:37, SEQID 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, SEQID NO:49, SEQ ID NO:50, SEQ IDNO: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 IDNO: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 listed sequence among the SEQ ID NO:78.

162. the method for claim 38, wherein, one of them capture oligo comprises SEQID 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 IDNO: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 IDNO: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, SEQID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ IDNO:59, SEQ ID NO:60, SEQID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQID 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 listed sequence among the SEQID NO:78.

163. the method for claim 38, wherein, one of them target nucleic acid sequence is the part of aureus gene, and one of them target nucleic acid sequence is the part of mecA gene.

164. the method for claim 38, wherein this method is used for distinguishing same two or more kinds of genus.

165. the method for claim 164, wherein said kind has the difference of two or more discontinuous kernel thuja acids.

166. the method for claim 164, wherein said kind has the difference of two or more continuous nucleotides.

167. the method for claim 164, wherein said kind has the difference of at least one Nucleotide.

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