CN101191143A - Gene chip without nucleic acid marking and its detecting method and application - Google Patents

Abstract

The invention discloses a gene chip without necessity of nucleic acid markings, comprising a DNA-RNA-DNA probe fixed on a solid-phase carrier, wherein, a DNA segment on one end of the probe is marked, and a connecting arm is arranged on the tail of a DNA segment on the other end of the probe. The invention also discloses a test method by utilization of the gene chip and application of the chip in clinical diagnostic tests. The gene chip of the invention has the advantages of capability of making genome DNA easier to directly perform chip tests, great improvement of detectable rate of low expression genes, higher flux level and lower cost.

Description

Need not gene chip and the detection method and the application of nucleic acid marking

Technical field

The present invention relates to a kind of gene chip, relate in particular to a kind of gene chip that need not nucleic acid marking; In addition, the invention still further relates to the detection method and the application of this gene chip.

Background technology

Gene chip is the scientific payoffs of the tool using value brought of the Human Genome Project, and it has merged multi-disciplinary state-of-the-art technologies such as life science, chemistry, microelectronics, computer science, statistics and life-information.Along with the development of micro-processing technology, the high-density of high density oligonucleotide chip can reach up to a million probes, so the gene chip flux level is not subjected to chip technology restriction itself, but is subjected to the processing of sample and the restriction of gene chip method of design.At human genome, common single nucleotide polymorphism (single nucleotide polymorphism, SNP) site surpasses 1,000 ten thousand, accounts for 90% (the The International HapMap Consortium.The International HapMap Project.Nature.2003 in total SNP site; 426:789-96).Though haplotype (haplotype) can be measured via a few label (tag) SNPs, but the assignment of genes gene mapping is carried out in the common variation between individuality approximately needed a hundreds of thousands of label SNPs, different (the Gabriel SB of its number with different colonies, Schaffner SF, Nguyen H, et al.The structure of haplotype blocks in the humangenome.Science.2002; 296:2225-9; Stephens JC, Schneider JA, Tanguay DA, et al.Haplotype variation and linkage disequilibrium in 313 human genes.Science.2001; 293:489-93).Therefore by the variation of causing a disease of linkage disequilibrium (linkage disequilibrium) location, even conservative estimation, the quantity of the required SNP genetic marker of association study (association study) is about hundreds of thousands of comprehensively.Although the SNP chip list primer amplification technology of Affymetrix company is owing to limit by restriction enzyme site, can not detect and be arranged in length and the SNPs of short restriction fragment, but the multiple level of this technology single tube reaction can reach 1～250,000 (Matsuzaki H, Loi H, Dong S, et al.Parallelgenotyping of over 10,000 SNPs using a one-primer assay on a high-densityoligonucleotide array.Genome Res.2004; 14:414-25; Matsuzaki H, Dong S, LoiH, et al.Genotyping over 100,000 SNPs on a pair of oligonucleotide arrays.Nat Methods.2004; 1:109-11), thereby SNP quantity can remedy its quantity of information defect of insufficient to a certain extent, is current unique SNP typing method that can reach full genome association study substantially.Yet single primer amplification technology can not expressly be analyzed selected SNPs, is not suitable for custom chip, is difficult to be applied to the clinical detection diagnosis.This also is reflected at the AmpliChip CYP450 of itself and Roche Holding Ag cooperative development, this chip only contains CYP2D6 and some common genotype of two genes of CYP2C19, in other important drug metabolism enzyme gene such as CYP3A4 all is not included in, and employing multiple PCR technique amplification target DNA fragment (Chou WH, Yan FX, Robbins-WeilertDK, et al.Comparison of two CYP2D6 genotyping methods and assessment ofgenotype-phenotype relationships.Clin Chem.2003; 49:542-51).Other high-throughout amplification technique has GoldenGate, molecular inversion probes (molecular inversion probes) and multiplex PCR etc., the multiple level of single tube reaction is about about 2000, but owing to be subjected to target DNA to the competition of primer and the problems such as reaction between the primer, the room for promotion of the multiple level of these technology is obviously very limited, and DNA is processed into the bottleneck place for the SNP chip.The application of high-throughput SNP chip in clinical diagnosis still depends on the development of aspects such as DNA processing, chip design.

Directly carry out the SNP somatotype and obviously can avoid this problem of DNA cloning, and can carry out somatotype to millions of SNPs simultaneously with genomic dna.The genome complicacy of unicellular lower eukaryote is lower, successfully utilize chip directly genomic dna to be carried out polymorphic analysis, these biologies comprise yeast (genomic dna is about 12Mb) (Winzeler EA, Richards DR, Conway AR, et al.Direct allelic variation scanning of the yeastgenome.Science.1998; 281:1194-7) and Arabidopis thaliana (genomic dna is about 120Mb) (BorevitzJO, LiangD, PlouffeD, et al.Large-scale identification of single featurepolymorphisms in complex genomes.Genome Res.2003; 13:513-23).Yet, though the human genome DNA successfully is applied to cDNA, BAC and oligonucleotide chip comparative genome hybridization (comparative genomic hybridization, CGH) chip (Ishkanian AS, Malloff CA, Watson SK, et al.A tiling resolution DNA microarray with complete coverageof the human genome.Nat Genet.2004; 36:299-303), but because human genome DNA's high complexity, the difficulty of directly carrying out snp analysis obviously will be higher than unicellular lower eukaryote, differentiates that from about 3,000,000,000 base-pair sequences a SNP is still an arduous challenge job.By research in recent years, existing several technology can be carried out the SNP somatotype to the human genome DNA, comprise the Radioactive colloidal gold detection technique, invade cutting (invasive cleavage), invade cutting in conjunction with technology such as rolling circle amplification (rolling circle amplification) and allele-specific primers extension method (allele-specific primer extension) binding signal cascade amplifications, but these technology ubiquities DNA consumption is big, signal to noise ratio is low, shortcomings such as complicated operation or low multiple level, their application aspect field such as disease gene group and pharmacogenomics and clinical diagnosis still await further development and perfect.On the other hand, human genome has ten thousand genes of 2-2.5 approximately, though it is simple for present chip of expression spectrum technology to contain all genes of these human genomes and expressed sequence tag, but often can't detect the low gene of expression level, also can't solve even use RNA linear amplification technology.Improve probe length and can improve recall rate to a certain extent, yet when probe length increased, the specificity of the local sequence of probe also greatly reduced, thereby causes false positive rate greatly to increase.The low low problem of expressing gene recall rate is one of current chip of expression spectrum urgent problem.

In view of nucleic acid amplification technologies is not enough to solve the problem of gene expression profile and the existence of SNP chip, how amplifying fluorescent signal will be the key point that improves the chip detection ability.Cascade is amplified because magnification is limited, obviously not as invading cutting, this genomic dna that also is reflected at based on these two kinds of technology directly carries out used DNA amount (the Gunderson KL of SNP somatotype, Steemers FJ, Lee G, et al.A genome-wide scalable SNPgenotyping assay using microarray technology.Nat Genet.2005; 37:549-54).Invading the detection of cutting the target DNA sequence is that (fluorescence resonanceenergy transfer FRET) realizes by FRET (fluorescence resonance energy transfer).So-called FRET transfers to contiguous another acceptor fluorescence group or quenching group with the non-reflectivity of energy after the irradiation of donor fluorophor stimulated luminescence, making the latter energy be discharged or be transformed into heat energy with himself distinctive wavelength of transmitted light comes out, whether its transmission efficient and two fluorescence are in maximum emission-absorption spectrum ranges and between the two distance dependent (Ozaki H, McLaughlin LW.The estimationof distances between specific backbone-labeled sites in DNA using fluorescenceresonance energy transfer.Nucleic Acids Res.1992; 20:5205-14).Invading cutting is according to aileron endonuclease (flap endonucleases, FEN) characteristic of cutting enzyme designs an intrusion probe and two SNP allele-specific probes, pleomorphism site is placed overlapping (the Olivier M.The Invader assay for SNP genotyping.Mutat Res.2005 that invades between probe and the allele-specific probe; 573:103-10).5 ' of allele-specific probe end is just as under the intrusion of invading probe and be wing and lean out like this, and then downcut by FEN, causes the fluorophor of 5 ' end and inner quenching group to separate, thus the generation fluorescent signal.In case after the allele-specific probe of hybridization was cut by FEN, after residual probe fragment came off and leaves, new uncut allele-specific probe can be attached on the same site again, thereby reached the purpose that signal amplifies.This design is in 90 minutes, the cutting rate of each target DNA sequence is greatly about 3000 probe molecule (Lyamichev V, Mast AL, Hall JG, et al.Polymorphism identification andquantitative detection of genomic DNA by invasive cleavage of oligonucleotideprobes.Nat Biotechnol.1999; 17:292-6), the signal amplification makes genomic dna can directly carry out the SNP somatotype without amplification efficiently, but maximum defective is that signal to noise ratio is low, and probe design is comparatively complicated, and its final application in the SNP chip still awaits further developing and be perfect.

Circle probe technology (cycling probe technology, CPT) be another kind of technology (the Duck P that the target DNA signal is amplified, Alvarado-Urbina G, Burdick B, et al.Probe amplifier system basedon chimeric cycling oligonucleotides.Biotechniques.1990; 9:142-8).The CPT probe is one to contain the mosaic of DNA-RNA-DNA, is about 25-30 base, includes 4-6 successive purine nucleotides.Its principle is in isothermal reaction, and behind probe and the strand target DNA sequence hybridization, under the effect of ribonuclease H (RNase H), the RNA part in the DNA-RNA-DNA probe is cut by specificity.RNase H is a kind of endoribonuclease, and its hydrolysis specifically hybridizes to the RNA phosphodiester bond on the DNA chain, so can decompose the RNA chain in the RNA/DNA hybridization system, this enzyme can not digest single stranded RNA, strand or double-stranded DNA.Because the annealing temperature of the probe fragment after enzyme is cut is far below temperature of reaction, the two ends DNA part of the CPT probe after the cutting also splits away off from the target DNA sequence thereupon, make the target DNA sequence can combine with complete probe again, thereby improved the rate of utilization of target DNA sequence.Therefore, every target DNA sequence can combine with many complete probe hybridizations, thereby produces the probe fragment of a lot of cuttings, and its amount was piled up along with the time, was linear amplification.By attached gel electrophoresis or immunology detection analysis, CPT successfully is applied to bacterium and Drug Resistance Detection.Because CPT has the characteristic of linear amplification, fluorophor and quenching group on the two ends of probe dna sequence dna difference mark just can amplify fluorescent signal, reach the purpose that real-time quantitative detects.And RNase H exists sequence dependent to the cutting of DNA-RNA-DNA probe, have only the probe that RNA part and target DNA sequence are mated fully just can be cut, so CPT also can be used for the SNP detection.

Cutting is the same with invading, and the characteristics of CPT maximum are exactly the amplified reaction that does not need this chain type, and they are that signal to DNA or RNA increases.And owing to do not need the chain type amplification procedure,, do not need as PCR, to heat up repeatedly, lower the temperature, therefore can save a lot of times as long as entire reaction course is controlled at the annealing temperature of probe.And CPT is different with PCR, and they are not that the target DNA sequence of amplifying is detected, therefore can reduce because the false positive that the PCR self-pollution causes.Have only when special DNA exists, this method just can make the signal analysis accumulation, and available multiple instrument detecting.Than invading cutting, CPT only designs a sequence-specific probe or two allele-specific probes, designs comparatively simply, and the gene order that can detect is more.On the other hand, in invading cutting, can only lack a base with target DNA sequence complementary sequence length in the probe fragment after enzyme is cut than complete probe, probe fragment and complete probe and the annealing and the isolating kinetics basically identical of target DNA sequence, both exist competition with combining of target DNA sequence.And the CPT probe is with after the target DNA sequence combines, partly cut from the centre by RNase H, the annealing temperature of the probe fragment after enzyme is cut is far below temperature of reaction, and the probe fragment that comes off unlikely is attached on the target DNA sequence again, and this makes the signal scale effect of CPT cut far above invading.Therefore, if CPT is applied in the gene chip, the signal to noise ratio of generation will obviously be better than invading cutting, can significantly improve the recall rate of low expressing gene, and reduce the difficulty that genomic dna directly carries out the SNP somatotype, be applicable to high-throughput, large-scale detection of nucleic acids more.

The technology of current SNP chip and chip of expression spectrum is comparatively perfect, but it is required far can not to satisfy the diagnosis of biomedical research and clinical detection.Under the situation that does not influence accuracy rate, how to improve the low expression of gene spectrum chip recall rate of expression level, how genomic dna is directly applied to the SNP chip detection with minimum amount, be the difficult point place of current biochip technology always.Can improve fluorescent signal to a certain extent though nucleic acid amplification or cascade are amplified, these technology can not essence address these problems, and operation is comparatively complicated.

Summary of the invention

One of the technical problem to be solved in the present invention provides a kind of gene chip that need not nucleic acid marking, and this chip is the amplification detection signal effectively, thereby significantly improves the recall rate of the low gene of expression level in the chip of expression spectrum.

Two of the technical problem to be solved in the present invention provides a kind of method that the said gene chip detects of using.

Three of the technical problem to be solved in the present invention provides the application of said gene chip in clinical diagnosis detects.

In order to solve the problems of the technologies described above, the present invention is achieved through the following technical solutions:

In one aspect of the invention, provide a kind of gene chip that need not nucleic acid marking, having comprised: be fixed on the DNA-RNA-DNA probe on the solid phase carrier, the dna fragmentation of described probe one end is carried out mark, and the dna fragmentation end of the other end can be provided with connecting arm.

Described mark comprises: fluorescein-labelled, biotin labeling, radioelement mark and enzyme labelling.

Described connecting arm passes through its terminal link molecule stationary probe on solid phase carrier.

Solid-phase matrix described in the present invention can be selected the known matrix in field for use, as long as described matrix is compatible with described reactant, it is just passable can not influence detected result.

The sequence of described probe and the complementation of target DNA sequence, the RNA sequence of preferred described probe and target DNA sequence are complementary fully, and the continuous complementary base radix of the RNA sequence of this probe and any sequence of probe is no more than 3.

The annealing temperature of described probe is 37～75 ℃, and the annealing temperature of the dna sequence dna at described probe two ends is hanged down 10 ℃ at least than the annealing temperature of probe.

Described probe sequence can comprise 1～10 base mismatch, preferably, can comprise 1～5 base mismatch, more preferably, can comprise 1～2 base mismatch.

Gene chip of the present invention also comprises at least a contrast probe, and described contrast probe is selected from: negative control probe, positive control probe and immobilization contrast probe.

Described probe can be fixed on the solid phase carrier by connecting arm.Connecting arm can provide one the space is sterically hindered to reduce freely for probe forms double-stranded part, carrying out [the Afanassiev V that helps hybridization, HanemannV, Wolfl S.Preparation of DNA and protein micro arrays on glass slidescoated with an agarose film.Nucleic Acids Res.2000,28:e66; USA Patent No.5556752].Connecting arm is long more, and hybridization efficiency is high more.Typical connecting arm comprises 15～30 functional group length.Connecting arm can be selected the functional group of appropriate form for use, as the mosaic of Poly T (A, C or G), C atom or polyethylene glycol and Poly T (A, C or G), poly-ethanol, poly-cruel, poly-ammonia, cruel and its composition of poly-sulfuric acid.

Described probe or connecting arm are fixed on the solid phase carrier by link molecule.Probe stationary can be passed through the realization of C-C key to carrier, for example, and the voltalef surface; Or better use siloxane bond (glass or silicon-dioxide use when making upholder).The siloxane bond bonding can be by upholder and link molecule Trichloromonosilane base or radical reaction such as trialkoxysilyl finish.Aminoalkyl group silane, light basic alkyl silane, 2 one light ethyl one aminopropyl triethoxysilanes, light ethyl one aminopropyl triethoxysilane or light propyl-triethoxysilicane all are surface adsorption groups of great use.

Described probe can be modified, and modifying method can be 5 '-NH2 modification, 5 '-SH modification, 5 '-Poly T (A, C or G) modification, 5 '-biotin modification, 3 '-NH2 modification, 3 '-SH modification, 3 '-Poly T (A, C or G) modification and 3 '-biotin modification etc.

In another aspect of this invention, provide a kind of method that the said gene chip detects of using, comprised the steps: 1) extracting testing sample nucleic acid; 2) choose the said gene chip; 3) be suitable under the condition of hybridizing, adding sample nucleic acid and contain the hybridization solution of RNase H enzyme, and make it react the enough time with selected gene chip; 4) result of hybridization is detected in the washing back.

RNase H enzyme among the present invention comprises high temperature resistant and non-refractory RNase H enzyme, and high temperature resistant RNase H enzyme all can add before and after sex change, and non-refractory RNase H enzyme adds in sex change cooling back.

Described hybridization temperature is 25 ℃～72 ℃, and described hybridization time is 1 minute～18 hours.Can change rigorous degree, the hybridization specificity of hybridization conditions to improve or to reduce hybridization.

The washing of described results of hybridization before detection can be used any suitable washings, and this washings can contain the tensio-active agent of 0～3% (w/w), washs sustainable reasonable time, as 1～30 minute.Can wash with the washings of room temperature, or the preheating after scouring, as 42 ℃.Available different washings successively washs.

In another aspect of this invention, also provide the application of gene chip of the present invention in clinical diagnosis detects.

Gene chip of the present invention makes sample of nucleic acid not need any fluorescent mark promptly to can be used for chip hybridization and detects, the detectivity of gene chip is reached a new height, not only reduce the chip detection difficulty that genomic dna directly carries out aspects such as SNP, and significantly improve the recall rate of the low gene of expression level in the chip of expression spectrum, in addition, because need not carry out loaded down with trivial details nucleic acid handles, and used probe mark fluorophor on the dna fragmentation of free end only, and do not add any quenching group, gene chip operation of the present invention is simpler, flux level is higher, and expense is also cheaper.

Description of drawings

Fig. 1 is the schematic diagram that chip hybridization of the present invention and signal amplify;

Fig. 2 is the chip hybridization figure as a result of the embodiment of the invention 2;

Fig. 3 is the chip hybridization figure as a result of the embodiment of the invention 4;

Fig. 4 is the chip hybridization figure as a result of the embodiment of the invention 5.

Embodiment

The present invention is further detailed explanation below in conjunction with drawings and Examples.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.

The preparation of embodiment 1 probe design and gene chip

1. probe design: the standard of probe is as follows: 1) sequence of selected gene or SNP site flank is got rid of the gene order or the SNPs of sequence-specific difference, in order to avoid cause non-specific hybridization through Blast; 2) the SNP site is in the RNA sequence, and is positioned at the middle portion of probe; 3) between probe annealing temperature 48-60 ℃, the annealing temperature of two ends DNA part is than low 15 ℃ at least of whole piece probes, and the probe fragment after enzyme is cut separates with the target DNA sequence; 4) probe self complementary sequence is no more than 3 base pairs, and must not form dimer between the probe, and RNA sequence particularly is in order to avoid produce high fluorescence background; 5) carry out SNP Blast, guarantee that probe sequence outside purpose SNP site, does not have other polymorphic sites, in order to avoid influence hybridization efficiency.

2. the preparation of chip:

(1) probe dissolving

Probe is synthetic, and 5 ' end at probe adds the preceding paragraph connecting arm earlier, and again with every probe TE solution dilution, final concentration is 10mM.With concentration be the probe of 10mM and PBS solution that concentration is 200mM in the medium volume mixture of 384 orifice plates, seal 384 orifice plates with adhesive sheet, vibration is 2 minutes under the room temperature, and is centrifugal ,-20 ℃ of preservations are used in order to point sample.

(2) point sample

The probe that designs and synthesizes in advance is downloaded on the solid phase carrier sheet base of materials such as slide, silicon chip by contact point sample or ink jet type point of sample, sets up the positive and negative control simultaneously.The sheet base adopts Cell Associates CSS-100 aldehyde radical sheet base, the point sample instrument of Ominigrid 100 models of GeneMachine company, humidity: 65-75% (being as the criterion) with FullMoon sheet base, temperature is a point sample under 25 ℃ the condition, and the form of point sample is 1 * 3, and each matrix is 8 * 18, after point sample finishes, after placing half an hour, chip is taken out, drying at room temperature is preserved.

Embodiment 2

1. goal gene amplification

(1) primer sequence:

Primer 1F 5 '-gtcgcaacctggtgcctataa-3 ' (SEQ ID NO:1);

Primer 1R 5 '-tgctataagccagctgagagattt-3 ' (SEQ ID NO:2);

Primer 2 F 5 '-aagccaaggctatgacattct-3 ' (SEQ ID NO:3);

Primer 2 R 5 '-aattcccggagaacttgtgct-3 ' (SEQ ID NO:4).

(2) probe sequence: CY3 is a fluorophor, and the capitalization base sequence is the RNA sequence in the center bracket

rs13431727-A

5’-NH2-C18-ggcctt(ATAG)gggaattta(Cy3)-3’(SEQ?ID?NO：5)；

rs13431727-T

5’-NH2-C18-ggcctta(TTGG)ggaattta(Cy3)-3’(SEQ?ID?NO：6)；

rs13431727-C

5’-NH2--C18-ggcctta(TCGG)ggaattta(Cy3)-3’(SEQID?NO：7)；

rs1146808-A

5’-NH2-C18-ggaaatgt(TATC)aaattatctg(Cy3)-3’(SEQ?ID?NO：8)；

rs1146808-C

5’-NH2--C18-ggaaatgt(TCTC)aaattatct(Cy3)-3’(SEQ?ID?NO：9)；

rs1146808-C

5’-NH2--C18-ggaaatgt(TGTC)aaattatct(Cy3)-3’(SEQ?ID?NO：10)。

Primer with sequence shown in SEQ ID NO:1～4 carries out pcr amplification, carries out in 60 μ l reaction systems, and reaction system is 0.3mM dNTP, 10mM Tris-HCl, 50mM KCl, 2mM MgCl ₂, 20%Q solution (Qiagen), upstream and downstream primer concentration 0.16 μ M, genomic dna 60ng, Taq enzyme 1.2U (Takara).Use Touch-down PCR response procedures [Don RH, Cox PT, Wainwright BJ, Baker K, MattickJS. ' Touchdown ' PCR to circumvent spurious priming during gene amplification.Nucleic Acids Res.1991,19:4008]: 94 ℃ of sex change 5mins; 94 ℃ of sex change 40s, 64 ℃ of annealing 1min, each circulation reduces by 0.5 ℃, and 72 ℃ are extended 30s, totally 10 circulations; 94 ℃ of sex change 40s then, 59 ℃ of annealing 40s, 72 ℃ are extended 30s, totally 30 circulations; Last 72 ℃ are extended 5mins.

2.PCR the product single stranded is handled

Get above-mentioned PCR product 5 μ l, increase, reaction system contains 0.3mM dNTP, 10mM Tris-HCl, 50mM KCl, 2mM MgCl ₂, 20%Q solution (Qiagen), downstream primer concentration 0.16 μ M and Taq enzyme 0.6U (Takara).PCR loop parameter: 94 ℃ of sex change 5min; 94 ℃ of sex change 40s then, 56 ℃ of annealing 40s, 72 ℃ are extended 50s, totally 35 circulations.

PCR product QIAquick PCR Purification Kit (Qiagen, Cat.No.28106) purifying:

1) the damping fluid PB of 5 times of PCR product volumes of adding, mixing need not to remove paraffin oil or kerosene.

2) the centrifugal post of QIAquick being positioned over the 2ml that test kit provides collects in the pipe.

3) sample is added in the centrifugal post of QIAquick, centrifugal 30-60s is with in conjunction with DNA.

4) outwell 2ml and collect centrifugal liquid in the pipe, the centrifugal post of QIAquick is put in the former collection pipe.

5) 0.75ml damping fluid PE is joined in the centrifugal post of QIAquick wash centrifugal 30-60s.

6) outwell 2ml and collect centrifugal liquid in the pipe, the centrifugal post of QIAquick is put in the former collection pipe centrifugal 1min.

7) the centrifugal post of QIAquick is positioned in the centrifugal pipe of autoclaved 1.5ml.

8) add 50 μ l damping fluid EB (10mM Tris-Cl, pH 8.5) or H in QIAquick film central authorities ₂O is with eluted dna, centrifugal 1min.In addition,, also can add 30 μ l elution buffers, leave standstill 1min in QIAquick film central authorities in order to improve the concentration of DNA, centrifugal then.

3. hybridization and washing

The principle that chip hybridization and signal reduce as shown in Figure 1, when RNase H partly cuts probe from middle RNA, be marked with 3 ' end dna fragmentation of fluorophor and be fixed on the end of 5 ' on solid phase carrier dna fragmentation and just come off from the target DNA sequence respectively, can remove fluorophor by washing, the fluorescent signal of chip is reduced.

The concrete steps of chip hybridization are that 95 ℃ of sex change of PCR product 10 minutes place on ice immediately, add the RNaseH mixing, are used for hybridization.Hybridization system: 275mM KCl, 50mM Tris-HCl (pH 8.3@25 ℃), 3mMMgCl ₂, 10mM DTT, 0.05%SDS, 15U RNase H, 200ng purified pcr product.60 ℃ of temperature were bathed 240 minutes.With containing 0.5 * SSC and 0.1%SDS washing lotion washing 5 minutes, use ddH at last then ₂O washing 15 seconds.

Chip after the washing, after drying, promptly available laser scanner scans (usefulness is GenePix4000B confocal laser scanner, also can with other laser scanner) here.The results of hybridization that chip after the scanning hybridization obtains is handled image with GenePix Pro again and is obtained data file as shown in Figure 2, the data file is just analyzed can be judged aim sequence then.

Embodiment 3

1. asymmetric PCR

Utilize primer and the probe sequence of embodiment 2 to carry out the asymmetric PCR amplification, carry out in 60 μ l reaction systems, reaction system is 0.3mM dNTP, 10mM Tris-HCl, 50mM KCl, 2mM MgCl ₂, 20%Q solution (Qiagen), the concentration 0.16 μ M of upstream primer, concentration 0.008 μ M, the genomic dna 60ng of upstream primer, Taq enzyme 0.6U (Takara).Use Touch-down PCR response procedures [Don RH, Cox PT, WainwrightBJ, Baker K, Mattick JS. ' Touchdown ' PCR to circumvent spurious priming duringgene amplification.Nucleic Acids Res.1991,19:4008]: 94 ℃ of sex change 5min; 94 ℃ of sex change 40s, 64 ℃ of annealing 1min, each circulation reduces by 0.5 ℃, and 72 ℃ are extended 50s, totally 10 circulations; 94 ℃ of sex change 40s then, 59 ℃ of annealing 40s, 72 ℃ are extended 40s, totally 30 circulations; Last 72 ℃ are extended 5min.(embodiment 2 described steps are pressed in operation to the PCR product for Qiagen, Cat.No.28106) purifying with QIAquick PCR Purification Kit.

2. hybridization and washing

95 ℃ of sex change of PCR product 10 minutes place on ice immediately, add RNase H mixing, are used for hybridization.Hybridization system: 275mM KCl, 50mM Tris-HCl (pH 8.3@25 ℃), 3mM MgCl ₂, 10mM DTT, 0.05%SDS, 15U RNase H, 200ng purified pcr product.60 ℃ of temperature were bathed 240 minutes.With containing 0.5 * SSC and 0.1%SDS washing lotion washing 5 minutes, use ddH at last then ₂O washing 15 seconds.

Chip after the washing, after drying, promptly available laser scanner scans (usefulness is GenePix4000B confocal laser scanner, also can with other laser scanner) here.The results of hybridization figure that chip after the scanning hybridization obtains handles image with GenePix Pro again and obtains data file, the data file is just analyzed can be judged aim sequence then.

Embodiment 4

1. total RNA extracting (TRIzol method)

(1) the 100mg parathyroid tissue can add 1ml Trizol, fully smashes tissue block with liquid nitrogen grinding or employing electric homogenizer.

(2) chloroform of about 1/5 volume of adding turned upside down abundant mixing about 1 minute, left standstill under the room temperature 5 minutes.

(3) 4 ℃, 12,000rpm carefully changes supernatant liquor over to new 1.5ml centrifuge tube after centrifugal 15 minutes, adds isopyknic Virahol, puts upside down mixing gently, and room temperature left standstill 5 minutes.

(4) 4 ℃, 12000rpm removes supernatant after centrifugal 10 minutes, adds 70% ethanol of 2/5 volume in precipitation, and 4 ℃, 12000rpm centrifuge washing precipitation 15 minutes.

(5) remove supernatant, the precipitation room temperature is dried the abundant dissolution precipitation of water that the back adds an amount of no RNA enzyme, measures A ₂₆₀And A ₂₈₀Value.

2.cDNA the preparation of probe

(1) from-70 refrigerators, takes out RNA, at room temperature thaw, in 0.2ml PCR pipe, prepare reaction soln then.

Total RNA 5 μ g

Random primer 6 aggressiveness (5 μ g/ μ l) 1 μ l

10mM?dNTP?mix 1μl

The water X μ l (make up water to 12 μ l) of nuclease free

Cumulative volume 12 μ l

(2) 70 ℃ of pre-sex change 10min place cooled on ice 2min immediately, subsequently the preparation cDNA first chain synthesis reaction system in the PCR pipe

5×first-strand?buffer 4μl

0.1M?DTT 2μl

RNA inhibitor 1 μ l

Cumulative volume 19 μ l

Adding 1 μ l Superscript II (200U/ μ l) behind 42 ℃ of insulation 2min (Invitrogen, USA).

(3) 42 ℃ are incubated 2 hours, 70 ℃ of sex change 10min.

3. hybridization and washing

Probe sequence: CY3 is a fluorophor, and the capitalization base sequence is the RNA sequence in the center bracket

rs13431727-A

5’-NH2-C18-ggcctt(ATAG)gggaattta(Cy3)-3’(SEQ?ID?NO：5)；

rs13431727-T

5’-NH2-C18-ggcctta(TTGG)ggaattta(Cy3)-3’(SEQ?ID?NO：6)；

rs13431727-C

5’-NH2--C18-ggcctta(TCGG)ggaattta(Cy3)-3’(SEQID?NO：7)；

CDNA places on ice immediately in 95 ℃ of sex change 10 minutes, adds RNase H mixing, is used for hybridization.Hybridization system: 275mM KCl, 50mM Tris-HCl (pH 8.3@25 ℃), 3mM MgCl ₂, 10mM DTT, 0.05%SDS, 20U RNase H, 15 μ g cDNA.60 ℃ of temperature were bathed 360 minutes.With containing 0.5 * SSC and 0.1%SDS washing lotion washing 5 minutes, use ddH at last then ₂O washing 15 seconds.

Chip after the washing, after drying, promptly available laser scanner scans (usefulness is GenePix4000B confocal laser scanner, also can with other laser scanner) here.The results of hybridization that chip after the scanning hybridization obtains is handled image with GenePix Pro again and is obtained data file as shown in Figure 3, the data file is just analyzed can be judged aim sequence then.

Embodiment 5DNA direct cross

Probe sequence: CY3 is a fluorophor, and the capitalization base sequence is the RNA sequence in the center bracket

bla2?5’-NH2-C18-cttgtcga(TTCTT)Cttgggat(Cy3)(SEQ?ID?NO：11)

blaZ?5’-NH2-C18-gccaagag(GTAAT)Gaaggaa(Cy3)(SEQ?ID?NO：12)

EH10?5’-NH2-C18-ccatctcg(AAAAA)Acggtgaa(Cy3)(SEQ?ID?NO：13)

EH1 5’-NH2-C18-tcagtaccta(AAAGA)Tattcagct(Cy3)(SEQ?ID?NO：14)

Extractive DNA carries out fragmentation with DNase I, and reaction system comprises:

37 ℃ of temperature are bathed 5min, 95 ℃ of 15min then.

95 ℃ of sex change of fragmentation product 10 minutes place on ice immediately, add RNase H mixing, are used for hybridization.Hybridization system: 275mM KCl, 50mM Tris-HCl (pH 8.3@25 ℃), 3mM MgCl ₂, 10mM DTT, 0.05%SDS, 10U RNase H.50 ℃ of temperature were bathed 120 minutes.With containing 0.5 * SSC and 0.1%SDS washing lotion washing 5 minutes, use ddH at last then ₂O washing 15 seconds.

Chip after the washing, after drying, promptly available laser scanner scans (usefulness is GenePix4000B confocal laser scanner, also can with other laser scanner) here.The results of hybridization that chip after the scanning hybridization obtains is handled image with GenePix Pro again and is obtained data file as shown in Figure 4, the data file is just analyzed can be judged aim sequence then.

Sequence table

＜110〉Shanghai Biochip Co., Ltd

＜120〉need not gene chip and the detection method and the application of nucleic acid marking

<130>NP-11068

<160>14

<170>PatentIn?version?3.3

<210>1

<211>21

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>1

gtcgcaacct?ggtgcctata?a 21

<210>2

<211>24

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>2

tgctataagc?cagctgagag?attt 24

<210>3

<211>21

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>3

aagccaaggc?tatgacattc?t 21

<210>4

<211>21

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>4

aattcccgga?gaacttgtgc?t 21

<210>5

<211>19

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(19)

<220>

<221>modified_base

<222>(19)..(19)

<400>5

ggccttatag?gggaattta 19

<210>6

<211>19

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(19)

<220>

<221>modified_base

<222>(19)..(19)

<400>6

ggccttattg?gggaattta 19

<210>7

<211>19

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(19)

<220>

<221>modified_base

<222>(19)..(19)

<400>7

ggccttatcg?gggaattta 19

<210>8

<211>22

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(22)

<220>

<221>modified_base

<222>(22)..(22)

<400>8

ggaaatgtta?tcaaattatc?tg 22

<210>9

<211>21

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(21)

<220>

<221>modified_base

<222>(21)..(21)

<400>9

ggaaatgttc?tcaaattatc?t 21

<210>10

<211>21

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(21)

<220>

<221>modified_base

<222>(21)..(21)

<400>10

ggaaatgttg?tcaaattatc?t 21

<210>11

<211>21

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(21)

<220>

<221>modified_base

<222>(21)..(21)

<400>11

cttgtcgatt?cttcttggga?t 21

<210>12

<211>20

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(20)

<220>

<221>modified_base

<222>(20)..(20)

<400>12

gccaagaggt?aatgaaggaa 20

<210>13

<211>21

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(21)

<220>

<221>modified_base

<222>(21)..(21)

<400>13

ccatctcgaa?aaaacggtga?a 21

<210>14

<211>24

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(24)

<220>

<221>modified_base

<222>(24)..(24)

<400>14

tcagtaccta?aaagatattc?agct 24

Claims (10)

1. gene chip that need not nucleic acid marking, comprising: be fixed on the DNA-RNA-DNA probe on the solid phase carrier, it is characterized in that, the dna fragmentation of described probe one end is carried out mark, and the dna fragmentation end of the other end can be provided with connecting arm.

2. gene chip as claimed in claim 1 is characterized in that, described mark comprises: fluorescein-labelled, biotin labeling, radioelement mark and enzyme labelling.

3. gene chip as claimed in claim 1 is characterized in that, described connecting arm passes through its terminal link molecule stationary probe on solid phase carrier.

4. gene chip as claimed in claim 1 is characterized in that, the annealing temperature of described probe is 37～75 ℃, and the annealing temperature of the dna sequence dna at described probe two ends is hanged down 10 ℃ at least than the annealing temperature of probe.

5. the method that application rights requires 1 described gene chip to detect is characterized in that, comprises the steps: 1) extracting testing sample nucleic acid; 2) choose the described gene chip of claim 1; 3) be suitable under the condition of hybridizing, adding sample nucleic acid and contain the hybridization solution of RNase H enzyme, and make it react the enough time with selected gene chip; 4) result of hybridization is detected in the washing back.

6. method as claimed in claim 5 is characterized in that, the described testing sample nucleic acid of step 1) is strand or double-stranded DNA or RNA.

7. method as claimed in claim 5 is characterized in that, the described sample nucleic acid of step 3) is strand or double-stranded DNA, preferred single stranded DNA.

8. method as claimed in claim 5 is characterized in that, hybridization temperature is 25 ℃～72 ℃ in the described step 3), and hybridization time is 1 minute～18 hours.

9. method as claimed in claim 5 is characterized in that, the described RNase H of step 3) enzyme comprises high temperature resistant and non-refractory RNase H enzyme.

10. the application of the described gene chip of claim 1 in clinical diagnosis detects.

Images