CN1824786A - Selective amplification process based on joining - Google Patents

Abstract

The present invention provides a method for amplifying object nucleotide sequence. Said method relates to polymerization and connection. It is characterized by that after the upstream oligonucleotide probe containing universal primer sequence, downstream oligonucleotide probe and target nucleic acid sequence are hybridized, the spacer sequence containing object nucleotide sequence between the above-mentioned two probes is filled up with polymerase, and the gaps of upstream oligonucleotide probe extended sequence 3' tail end and downstream oligonucleotide probe 5' tail end are closed by polymerase. Said invention uses connection product as template and utilizes universal primer to make PCR amplification. Said technique can be used for detecting DNA sequence change and expression spectrum.

Description

Based on the selective amplification process that connects

Technical field

The invention belongs to external nucleotide sequence amplification field, particularly a kind of archaeal dna polymerase and active method of dna ligase coexistence of depending at first with the amplification of mediation purpose nucleotide sequence.This method can be used for selective amplification and contains nucleotide sequence such as the sequence variations of point mutation, insertion and disappearance.

Technical background

Human genome is the height polymorphism, and it is different that 1% sequence is arranged in any two human individuals' the genome approximately.Different groups and individual in the difference aspect the susceptibility of disease and other biological character (as to the reactivity of medicine and sense of taste susceptibility etc.), its genetics basis is the variability of human genome DNA's sequence.The mutant dna sequence that several types is arranged in the human genome, these variations comprise the copy number difference of change, insertion, disappearance and the tumor-necrosis factor glycoproteins of single base.In human genome, content the most rich variations be single nucleotide polymorphism (Single nucleotide polymorphisms, SNPs).Single nucleotide polymorphism is meant and has different bases in the genome on the specific nucleotide position that the molecule that is formed by single nucleotide substitution, insertion or disappearance is polymorphic, is variation the most stable in the genome.Each base type on the same position is an allelotrope, and this position is called the SNP site.SNPs is modal a kind of in human heritable variation, accounts for more than 90% of all known polymorphisms.Any one colony's medium frequency is not less than nearly 1,000 ten thousand of 1% SNP site, accounts for 90% (the The International HapMap Consortium.The International HapMapProject.Nature.2003 Dec 18 in total SNP site; 426 (6968): 789-96.).SNPs has farthest represented the hereditary difference between the Different Individual, thereby becomes research multigenic disease, pharmacogenetics and human evolution's important genetic marker.

Developed multiple SNP typing method in more than ten years in the past, restricted property endonuclease digestion method commonly used, allele specific oligonucleotide hybridization, the mispairing of PCR primer, single-basic extension, oligonucleotide connect, tetra-sodium order-checking (pyrosequencing) and Invader^TMEtc. method.Directly no doubt can carry out the SNP somatotype with genomic dna, but because human genome DNA's height polymorphism, unique sequence only accounts for 45% of total DNA, this makes to have only those SNPs that are arranged in unique sequence directly to carry out somatotype with genomic dna, and those SNPs that are arranged in the higher sequence of tumor-necrosis factor glycoproteins or homology can not be by accurate somatotype.As have the CYP450 supergene family of vital role in drug metabolism, not only there is pseudogene in some CYP450 gene, and sequence homology is very high between the subtribe member.Therefore,, have only by after specific primer or this SNP place sequence of probe amplification for those SNPs that is arranged in the higher sequence of tumor-necrosis factor glycoproteins or homology, can be by somatotype accurately.Therefore, those directly carry out the SNP somatotype with genomic dna technology all exists SNP to select the low problem of degree of freedom.Except a few SNP typing methods such as DASH, most of SNP typing methods target DNA sequence that all needs in advance to increase.(Polymerase chain reaction PCR) can significantly improve the copy number of target DNA to polymerase chain reaction, thereby can effectively reduce the complicacy of aim sequence, and improves the somatotype accuracy rate.Traditional multiplex PCR a plurality of target DNA fragments that can increase simultaneously in same reaction tubes have improved the utilising efficiency that detects flux and DNA.5-10 the target DNA fragment yet most multiple PCR techniques can only increase, mainly be because of increase along with the primer number, the effectively difficult more control of reaction conditions of each target DNA fragment of amplification, and the reaction between primer and the primer has promoted the formation of primer dimer, consequently design of primers is loaded down with trivial details in the multiplex PCR, often is difficult to successfully in experiment.

The SNP chip that global cdna chip market occupation rate reaches the Affymetrix company of half is to adopt the single primer based on enzyme cutting method to detect, its principle is after utilizing the digestion with restriction enzyme genomic dna, each dna fragmentation is connected the joint that has the universal primer sequence, like this with regard to available universal primer amplification target DNA fragment (Matsuzaki H, Loi H, Dong S, Tsai YY, Fang J, Law J, Di X, Liu WM, Yang G, Liu G, Huang J, Kennedy GC, Ryder TB, Marcus GA, Walsh PS, Shriver MD, Puck JM, Jones KW, Mei R.Parallel genotyping of over10,000 SNPs using a one-primer assay on a high-density oligonucleotide array.Genome Res.2004Mar; 14 (3): 414-25.).The SNP chip that current flux and distribution density are the highest is exactly the Mapping500k Set of Affymetrix company, and individual chip promptly contains more than 250,000 SNP.Yet, though this technology flux is very high, owing to limit by restriction enzyme site, can't cuts the SNPs of dna fragmentation and analyze being arranged in longer enzyme, the selected SNP of somatotype optionally, SNP selection degree of freedom is lower.This also is reflected at the AmpliChip CYP450 gene chip of itself and Roche Holding Ag cooperative development, this chip only contains CYP2D6 and some comparatively common genotype of two genes of CYP2C19, in gene such as CYP2C9, CYP3A5 all is not included in, and adopt multiple PCR technique amplification target DNA fragment.

Existing SNP chip more is to use based on the selective amplification method that connects to handle DNA's, the ultimate principle of this method is to utilize dna ligase, specifically two adjacent on DNA chain oligonucleotide probes are connected, and allele-specific probe 3 ' terminal bases is unmatched, then can not be connected, can't increase, guarantee specificity.By on oligonucleotide probe, introducing the universal primer sequence, can after connection, use universal primer, be that template increases to connect product.And ligase enzyme mediation roll circular DNA amplification (rolling circle amplification, RCA) technology is by probe design being become a padlock-probe (circularizing padlock probe), here after padlock-probe and the target DNA fragment hybridization, 3 ' and 5 ' end is connected to form an annular DNA by dna ligase, makes the DNA cloning ability improve.Though improved multiple level by method based on the selective amplification that connects, but owing to do not have intervening sequence between back two oligonucleotide probes of hybridization, but it is closely adjacent, cause SNP to select degree of freedom still very limited, to directly to carry out the technology of SNP somatotype with genomic dna similar, and flux level is also comparatively limited.By fill up and two oligonucleotide probe complementary dna sequence dnas between the space be intervening sequence, the method that connects upstream oligonucleotide probe extension sequence 3 ' end and downstream oligonucleotide probe 5 ' end, not only improved the selection degree of freedom of SNP, and the also greatly raising of multiple level, can reach thousands of.By filling up the space of 1 base pair between padlock-probe two ends, its multiple level reaches as high as (Hardenbol P, Yu F up to ten thousand as molecular inversion probes method (molecular inversion probe), Belmont J, Mackenzie J, Bruckner C, Brundage T, Boudreau A, Chow S, Eberle J, Erbilgin A, Falkowski M, Fitzgerald R, Ghose S, Iartchouk O, Jain M, Karlin-Neumann G, Lu X, Miao X, Moore B, Moorhead M, Namsaraev E, Pastemak S, Prakash E, Tran K, Wang Z, Jones HB, Davis RW, Willis TD, Gibbs RA.Highly multiplexed molecularinversion probe genotyping:over 10,000 targeted SNPs genotyped in a single tube assay.GenomeRes.2005Feb; 15 (2): 269-75.).And current SNP selects the highest GoldenGate of degree of freedom^TMThe intervening sequence of technology is 1～20 base pair (Shen R, Fan JB, Campbell D, Chang W, Chen J, Doucet D, Yeakley J, Bibikova M, Wickham Garcia E, McBride C, Steemers F, Garcia F, Kermani BG, Gunderson K, Oliphant A.High-throughput SNP genotyping on universal bead arrays.Mutat Res.2005 Jun3; 573 (1-2): 70-82.).But the SNP of these technology selects degree of freedom still very limited, as in 1500 Quality Control SNPs of HapMap plan, has only 864 SNPs to be fit to use GoldenGate^TMDetect, SNP selects degree of freedom to have only 57.6% (Gunderson KL, Steemers FJ, Lee G, Mendoza LG, Chee MS.A genome-wide scalable SNPgenotyping assay using microarray technology.Nat Genet.2005 May; 37 (5): 549-54.).Therefore only will be not enough to increase substantially SNP selection degree of freedom with extending 1～20bp.

In view of current no matter be disease gene group, pharmacogenetics and pharmacogenomics or be clinical detection diagnosis, more investigator need analyze selected separately different SNPs, therefore needs a kind of SNP to select the bigger DNA treatment process of degree of freedom to be applied on the SNP typing method.The present invention replaces the conventional alleles-specific oligonucleotide probe (ASO) that uses with locus specificity oligonucleotide probe (LSO), and by increasing in the target nucleotide sequences and the intervening sequence between the upstream and downstream oligonucleotide probe complementary sequence, not only improve SNP greatly and select degree of freedom, and improved specificity.By increasing intervening sequence, adjoining polymorphic site can be increased by pair of L SO.By in LSO, introducing the universal primer sequence, can analyze a plurality of SNP simultaneously, reach high multiple level.And make an amendment slightly by oligonucleotide probe is designed, this technology equally also is applicable to gel electrophoresis and mass spectrometric detection system.This technology not only can be used for the detection of transgenation, in fact equally also can be used for the detection of chromosome abnormalty phenomenons such as gene insertion, disappearance; Can not only detect the change of dna sequence dna, and can be used for the detection of express spectra.

Summary of the invention

The purpose of this invention is to provide a kind of method of the purpose nucleotide sequence being carried out the specific selectivity amplification.

The invention provides a kind of method of the purpose nucleotide sequence that increases, it is characterized in that described target nucleotide sequences contains one section interested purpose nucleotide sequence.This method comprises that the sample nucleic acid molecule that hits is connected with another oligonucleotide probe with hybridization, the extension of oligonucleotide probe molecule, the oligonucleotide probe molecule extension sequence of oligonucleotide probe.Oligonucleotide probe contains one section and target nucleotide sequences complementary sequence and universal primer sequence.Form probe-target nucleotide sequences complex body behind target nucleotide sequences and the oligonucleotide probe hybridization.Under the effect of polysaccharase extend to the 5 ' end of with target nucleotide sequences 3 ' holding complementary downstream oligonucleotide probe with target nucleotide sequences 5 ' end complementary upstream oligonucleotide probe this moment.Ligase enzyme is with upstream oligonucleotide probe extension sequence 3 ' end and 5 ' the terminal connection of downstream oligonucleotide probe, and the result connects to contain one section and purpose nucleotide sequence complementary specific sequence in the product between the upstream and downstream oligonucleotide probe.Therefore utilize the universal primer that can discern universal primer sequence in the oligonucleotide probe, the purpose nucleotide sequence just can optionally be increased.

In an embodiment, the invention provides a kind of method of the purpose nucleotide sequence that increases, it is characterized in that described target nucleotide sequences contains one section interested purpose nucleotide sequence.Being connected of the extension, oligonucleotide probe molecule extension sequence that this method comprises hybridization, the oligonucleotide probe molecule of the fragmentation of sample double center chain nucleic acid molecule, the biotin labeling of fragmentation nucleic acid molecule, the immobilization of biotin labeled fragmentation nucleic acid molecule, immobilized nucleic acid molecule and oligonucleotide probe and another oligonucleotide probe, wash-out are collected and are connected product.Form probe-target nucleotide sequences complex body behind target nucleotide sequences and the oligonucleotide probe hybridization.Under the effect of polysaccharase extend to the 5 ' end of with target nucleotide sequences 3 ' holding complementary downstream oligonucleotide probe with target nucleotide sequences 5 ' end complementary upstream oligonucleotide probe this moment.Ligase enzyme is with upstream oligonucleotide probe extension sequence 3 ' end and 5 ' the terminal connection of downstream oligonucleotide probe, and the result connects to contain one section and purpose nucleotide sequence complementary specific sequence in the product between the upstream and downstream oligonucleotide probe.Therefore utilize the universal primer that can discern universal primer sequence in the oligonucleotide probe, the purpose nucleotide sequence just can optionally be increased.Contain one section and target nucleotide molecular moiety sequence complementary sequence in the oligonucleotide probe.

Method of the present invention also can be used for the situation that exists of detection specificity sequence, as point mutation, allelotrope disappearance or detections such as amplification, microorganism except that can be used for amplifying nucleic acid sequence.Therefore, except can detecting the target sequence that derives from human body, also can detect and come from other as target sequences such as animals and plants, microorganisms.

Description of drawings:

Fig. 1 is the selective amplification schema of purpose nucleotide sequences such as single target DNA sequence, polymorphic or mutational site.

Fig. 2 is the selective amplification schema of purpose nucleotide sequences such as a plurality of target DNA sequences, polymorphic or mutational site.

Fig. 3 is the selective amplification schema of integrating capture elution system.

Fig. 4 carries out schema polymorphic or that the mutational site is analyzed for the attached gel electrophoresis detection system.Two special allelotype oligonucleotide probes that contain the different lengths stuffer of design on polymorphic or mutational site, wherein 3 ' of oligonucleotide probe do not hold base and polymorphic or the mutational site is complementary, the length stuffer in the oligonucleotide probe is between universal primer sequence and target nucleic acid sequence complementary segment.Locus specificity oligonucleotide probe of design in the flanking sequence of the downstream in polymorphic or mutational site.After selectivity connects and increases, can utilize the detected through gel electrophoresis system to carry out the analysis in polymorphic or mutational site.

Fig. 5 carries out the schema of testing goal dna sequence dna for the attached gel electrophoresis detection system.In two flank sequences of target DNA sequence, respectively design an oligonucleotide probe, and introduce a segment length stuffer between the universal primer sequence of an oligonucleotide probe and the target nucleic acid sequence complementary segment therein, after selectivity connects and increases, can utilize the detected through gel electrophoresis system to detect.

Fig. 6 is for carrying out schema polymorphic or that the mutational site is analyzed in conjunction with the mass spectrum detection system.Two special allelotype oligonucleotide probes that contain the different mass stuffer of design on polymorphic or mutational site, wherein 3 ' of oligonucleotide probe do not hold base and polymorphic or the mutational site is complementary, the quality stuffer in the oligonucleotide probe is between universal primer sequence and target nucleic acid sequence complementary segment.Locus specificity oligonucleotide probe of design in the flanking sequence of the downstream in polymorphic or mutational site.After selectivity connects and increases, can utilize the mass spectrometric detection system to carry out the analysis in polymorphic or mutational site.

Fig. 7 is for carrying out the schema of testing goal dna sequence dna in conjunction with the mass spectrum detection system.In two flank sequences of target DNA sequence, respectively design an oligonucleotide probe, and introduce one section quality stuffer between the universal primer sequence of an oligonucleotide probe and the target nucleic acid sequence complementary segment therein, after selectivity connects and increases, can utilize the mass spectrometric detection system to detect.

Fig. 8 is the schema that is used for the selective amplification of genetic expression.

Embodiment

The present invention has developed a kind of method that is used for the amplification of purpose nucleotide sequence specific selectivity.

The method that the purpose nucleotide sequence is carried out specific selectivity amplification provided by the present invention comprises:

1) extends connection

Extend the formation of linked system: the DNA that contains the purpose nucleotide sequence is as template, a pair of oligonucleotide probe (oligonucleotide probe 5 ' the end phosphorylations of its middle and lower reaches), four kinds of triphosphate deoxyribose nucleotide (dATP, dCTP, dGTP, dTTP), dna ligase and archaeal dna polymerase, and suitable buffer system, comprise the cofactor (the needed NAD of Taq dna ligase) of dna ligase needs etc.Whole extension linked system is 90-99 ℃ of sex change after 3 to 10 minutes, carries out renaturation, extension more in succession and is connected.Heat-resistant dna ligase enzyme and hot resistant DNA polymerase can add before sex change in the lump, also can add after sex change, and heat labile dna ligase and archaeal dna polymerase needed to add after sex change again.Whole process comprises following three steps:

(a) sex change: template DNA is sex change in advance, or sex change after having added a series of reagent, makes the template DNA single stranded; Denaturation temperature is generally 90～99 ℃, and the time length is 1 second～10 minutes; Preferred temperature is 93～95 ℃, 2 minutes～5 minutes sex change time.

(b) renaturation: whole extension linked system is placed certain temperature, make in the upstream and downstream oligonucleotide probe can and specific nucleotide sequence complementary sequence and template between be suitable for mutual annealing, to form oligonucleotide probe-template DNA crossbred; This temperature is generally 25～72 ℃, 30 seconds～25 minutes time length; 45～65 ℃ of preferred temperature, 5～15 minutes time length.

(c) extend and is connected: in the temperature that suits, the upstream oligonucleotide probe 3 ' end that mates fully with template DNA in the described oligonucleotide probe of polymerase extension-template DNA crossbred is filled up the space between two oligonucleotide probes of upstream and downstream.Polysaccharase described here is not had strand displacement and a 5 prime excision enzyme activity, therefore after extending to downstream oligonucleotide probe 5 ' end, promptly comes off from template DNA.Breach between ligase enzyme sealing this moment upstream oligonucleotide probe extension sequence 3 ' end and downstream oligonucleotide probe 5 ' end.During this starting stage, connect and the polymerization coexistence.Contain the upstream and downstream sequence oligonucleotide probe and promptly can be used as template, utilize respectively and upstream and downstream oligonucleotide probe universal primer sequence complementary universal primer, just can increase on a large scale with the product that is connected with purpose nucleotide sequence complementary sequence.

2) pcr amplification

Described oligonucleotide probe and target nucleic acid in extension/linked system through extend with is connected after, remove the oligonucleotide probe that does not connect through exonuclease, or collect the connection product, or the not treated pcr amplification that directly carries out through catching elution system.The pcr amplification system constitutes: connect product, a pair of universal primer, four kinds of triphosphate deoxy-nucleotides (dATP, dCTP, dGTP, dTTP), heat-resisting polymerase, magnesium ion and suitable buffer system.Utilize universal primer, guaranteed that each connects product and can both be increased by equivalence.

The seizure elution system comprises: the biotinylation primer that catch with streptavidin or avidin (1); (2) product of the digoxigenin labeled of catching with anti digoxin antibody; (3) complementary oligonucleotide that adheres to magnetic bead or latex bead.

If one of four kinds of dNTPs have fluorescein, vitamin H, digoxin or isotropic substance, can amplify and have fluorescein, vitamin H, digoxin or isotope-labeled DNA.

If universal primer or Oligonucleolide primers contain the Nucleotide of modification or mark, then the Nucleotide of this modification or mark can be incorporated among the PCR product D NA that amplifies by fixed point.

Archaeal dna polymerase among the present invention and dna ligase can be cryogenic, also can be heat-stable.Except adding a kind of dna ligase, also can add the dna ligase mixture more than 2 kinds or 2 kinds in the reaction system.In reaction system, except adding a kind of archaeal dna polymerase, also can add the archaeal dna polymerase mixture more than 2 kinds or 2 kinds.

The present invention is the DNA cloning technology of a brand-new specific selectivity.After mending space between flat upstream and downstream oligonucleotide probe by the archaeal dna polymerase of high-efficiency polymerization ability in theory, seal breach with dna ligase again, and only extend and connect once, make each purpose nucleotide sequence amplification efficiency almost consistent.And follow-up PCR is general, expects that all products are nearly all increased by equivalence.

The present invention replaces alleles-specific oligonucleotide probe (ASO) with locus specificity oligonucleotide probe (LSO), by respectively design the LSO aim sequence that increases in the flanking sequence of purpose nucleotide sequences such as polymorphic site, mutational site or target dna sequence.Purpose nucleotide sequence to be detected is arranged in respectively the intervening sequence with two LSO complementary target nucleotide sequences.The length of intervening sequence depends on the specificity of purpose nucleotide sequence flanking sequence, if the flanking sequence specificity is low, and can be by the proper extension intervening sequence to guarantee the specificity of LSO sequence; Otherwise then intervening sequence is just short.Obviously, after upstream and downstream LSO and the hybridization of purpose fragment sequence, by polysaccharase fill up and upstream and downstream oligonucleotide probe complementary target nucleotide sequences between intervening sequence, just connect upstream oligonucleotide probe extension sequence 3 ' terminal and downstream oligonucleotide probe 5 ' end, rather than directly connect upstream oligonucleotide probe 3 ' terminal and downstream oligonucleotide probe 5 ' end by ligase enzyme.Owing to there is intervening sequence, the oligonucleotide probe scope of design enlarges, reduced the complicacy of oligonucleotide probe design on the one hand, improved the selection degree of freedom of purpose nucleotide sequence greatly, simultaneously also improve the specificity of oligonucleotide probe, thereby also guaranteed the specific amplification of purpose nucleotide sequence.SNP in the various in theory sequences all can be come out by amplification specifically, and high SNP selects degree of freedom to make this method be suitable for the research and the clinical detection diagnosis of every field such as disease gene group, pharmacogenetics fully.

The present invention replaces ASO amplification SNP sequence with LSO, not only improved SNP and selected degree of freedom, and intervening sequence can reach a hundreds of base pair, and adjoining SNPs can use in the lump with a pair of LSO, has reduced number of probes, thereby greatly reduces cost.By in LSO, introducing the universal primer sequence, after LSO and the DNA hybridization in succession after polysaccharase is filled up the breach that space between the upstream and downstream oligonucleotide probe and ligase enzyme sealing upstream oligonucleotide probe extension sequence 3 ' are terminal and downstream oligonucleotide probe 5 ' is terminal, connecting product with this is template, utilize universal primer just can carry out conventional pcr amplification, thereby guaranteed the high multiple level and the ease for operation of this method.This method provides the strong technique means that can carry out high speed, parallel acquisition and analysis to idiogenetics information in conjunction with the chip hybridization technology platform for the mankind.This technology not only can be used for the detection of transgenation, the detection of chromosome abnormalty phenomenon such as in fact equally also can be used for genetically deficient, repeat; Can not only detect the change of dna sequence dna, and can be used for the detection of express spectra.

Detection system of the present invention is compatible high, except can be applicable to the chip hybridization technology platform, does slightly to change also to can be applicable to other technology platform:

(1) detection system of PCR-based product length: by two ASOs probes of design and a LSO probe on each SNP site, and in every ASO, add different length stuffers (length stuffer) to distinguish different SNP allelotrope, maybe can design two ASOs padlock-probe.And non-polymorphism such as genetically deficient or sudden change detect for detecting, and only need design two LSO and get final product.Obviously, than SNPWave^TM(van Eijk MJ, Broekhof JL, van der PoelHJ, Hogers RC, Schneiders H, Kamerbeek J, Verstege E, van Aart JW, Geerlings H, Buntjer JB, vanOeveren AJ, Vos P.SNPWave:a flexible multiplexed SNP genotyping technology.Nucleic AcidsRes.2004 Mar 5; 32 (4): e47.) and MLPA (Schouten JP, McElgunn CJ, Waaijer R, Zwijnenburg D, Diepvens F, Pals G.Relative quantification of 40 nucleic acid sequences by multiplexligation-dependent probe amplification.Nucleic Acids Res.2002 Jun 15; 30 (12): technology such as e57.), the present invention is because the strategy of having taked first extension to connect again, substantially can be distinguished by the length of adjusting intervening sequence between different SNP site, therefore need not in probe, to introduce very long length stuffer, thereby improved specificity and multiple level, and reduced cost.Limited by cost and synthetic probe length, the multiple level of padlock-probe strategy of the present invention is only a little more than SNPWave^TMOne of advantage of this technology is the order-checking platform that detection system can be used current widespread use.

(2) based on mass spectral detection system: the present invention equally also is applicable to (matrix-assisted laser desorption/ionization time-of-flight of substance assistant laser desorpted ionized flight time, mass spectrum detection such as MALDI-TOF), this need make two places and revise in the design of LSO: i) stuffer among the LSO keeps uniform quality, rather than the length unanimity; But ii) in amplified production, introduce cutting fragment, make the detection lug section be positioned within the MALDI-TOF best in quality sensing range.The present invention can with the coexistence of many seizure and detection system, this makes that whole proposal can automatization, has improved flux, can the quick analysis great amount of samples.

Compare with existing DNA treatment process, this method has bigger advantage: easy and simple to handle, flux is big, good reproducibility, detection system are compatible high as a result, the accuracy, susceptibility and the specificity that detect have been guaranteed in conjunction with chip technology, and can select SNPs at random according to scientific research and clinical needs, SNP selects degree of freedom big.

Embodiment

1. genomic DNA fragmentization

Human gene group DNA 10 * DNase I reaction buffer DNase I (0.1U/ μ l) ddH2O

5μg(80μl) 10μl 1μl 9μl

37 ℃ of temperature are bathed 10min, 95 ℃ of 15min then.

2. the purifying of fragmentation genomic dna

DNase I fragmentation genomic dna 3M NaAc precooling dehydrated alcohol

100μl 10μl 320μl

Deposit 15min for-20 ℃, the centrifugal 15min of 12500rpm then, 75% alcohol washing 2 times, air-dry at last.

3. deoxynucleotidyl transferase 3 ' terminal biotin labeling

The DNase I fragmentation genomic dna of purifying (is dissolved in ddH2O) 5 * terminal deoxynucleotidyl transferase reaction buffer vitamin H-N6-ddCTP (0.4mM) terminal deoxynucleotidyl transferase (20U/ μ l)

37μl 10μl 1μl 2μl

37 ℃ of temperature are bathed 60min, then 95 ℃ of heating 15min.

4. the genomic dna of the plain mark of purifying biological

Biotin labeled genomic dna 3M NaAc precooling dehydrated alcohol

50μl 5μl 250μl

Deposit 15min for-20 ℃, the centrifugal 15min of 12500rpm then, 75% alcohol washing 2 times, air-dry at last.

5. biotin labeled genomic dna is fixed on the magnetic bead of avidin bag quilt.

Biotin labeled genomic dna is in 95 ℃ of sex change 5min.

Biotin labeled genomic dna (250ng/ μ l) SNP primer (50nM) 2 * binding buffer liquid ddH2O

4μl 10μl 25μl 11μl

55 ℃ of temperature are bathed 60min.Use 1 * binding buffer liquid then in succession, 1 * lavation buffer solution, each washing of 1 * extension/ligation damping fluid is once.

1 * binding buffer liquid: 5mM Tris-HCl (pH 7.5), 1.0M NaCl, 0.5mM EDTA.

1 * lavation buffer solution: 5mM Tris-HCl (pH 7.5), 1.0M NaCl, 0.5mM EDTA.

1 * extension/ligation damping fluid: 10mM Tris-HCl (pH 8.3), 10mM KCl, 2.5mM MgCl₂, 1mMNAD, 0.01% Triton X-100

6. be fixed in the extension/ligation on the magnetic bead of avidin bag quilt

Every pipe contains

10 * reaction buffer Stoffel fragment DNA polymerase (ABI) 5mM NAD Taq DNA Ligase (40U/ μ l) ddH2O

5μl 0.5μl 10μl 1μl 33.5μl

50 ℃ of temperature are bathed 60min, and every then effective 1 * lavation buffer solution washing is once used ddH at last₂The O washing once.

7. wash-out connects product

Every pipe adds 50 μ l ddH₂O in 95 ℃ of heating 4min, places cold cut on ice then immediately.Supernatant liquor is forwarded in another clean pipe.

8.PCR reaction

10 * reaction buffer 25mM MgCl210mM dNTPs 50pmol universal primer (T3 and T7) 5U/ μ l AmpliTaq Gold DNA polymerase (ABI) wash-out is connected product ddH2O

9μl 7.5μl 3μl 1.2μl 0.5μl 50μl 28.8μl

The loop parameter of using in the PCR reaction is as follows: 95 ℃ of 5min of initial sex change; 95 ℃ of 40sec of sex change, the 64 ℃ of 40sec that anneal extend 72 ℃ of 40sec, totally 40 circulations; 72 ℃ of 10min of final extension.After the circulation, reaction mixture is cooled to 4 ℃.

Claims (13)

1. method of purpose nucleotide sequence that increases, this method comprises:

(1) form polymerization-ligation mixture, comprising:

(i) target nucleic acid sequence;

(ii) oligonucleotide probe, comprise the upstream oligonucleotide probe, its 3 ' end has at least a part of first section with first end of target nucleic acid sequence complementary basically, the downstream oligonucleotide probe, its 5 ' end has at least a part of second section with second end of target nucleic acid sequence complementary basically, in the intervening sequence of purpose nucleotide sequence between first section and second section, wherein terminal the and downstream oligonucleotide probe of upstream oligonucleotide probe 5 ' 3 ' is terminal respectively contains one section universal primer sequence respectively;

(iii) at least 4 kinds of different nucleotide bases;

(iv) polysaccharase and ligase enzyme;

(2) utilize universal primer, the upstream oligonucleotide probe extension sequence that amplification has connected and the product that is connected of downstream oligonucleotide probe produce a large amount of amplicons;

(3) detect described amplicon, be present in the described sample to show described purpose nucleotide sequence.

2. the method for amplification in vitro purpose nucleotide sequence according to claim 1, it is characterized in that one of described four kinds of nucleotide bases have isotropic substance, vitamin H, digoxin or fluorescent mark, amplified production is to have isotropic substance, vitamin H, digoxin or fluorescently-labeled nucleotide sequence.

3. the method for amplification in vitro purpose nucleotide sequence according to claim 1, it is characterized in that one of described universal primer has isotropic substance, vitamin H, digoxin or fluorescent mark, amplified production is to have isotropic substance, vitamin H, digoxin or fluorescently-labeled nucleotide sequence.

4. the method for amplification in vitro purpose nucleotide sequence according to claim 1 is characterized in that described ligase enzyme or polysaccharase can be cryogenic, also can be heat-stable.

5. the method for amplification in vitro purpose nucleotide sequence according to claim 1 is characterized in that described ligase enzyme or polysaccharase can be a kind of dna ligase or polysaccharase, also can be dna ligase or polysaccharase mixture more than 2 kinds or 2 kinds.

6. the method for amplification in vitro purpose nucleotide sequence according to claim 1, it is characterized in that described oligonucleotide probe except the universal primer sequence and with target nucleotide sequences complementary sequence, can contain other sequences.

7. the method for amplification in vitro purpose nucleotide sequence according to claim 1 is characterized in that described intervening sequence is polymorphic site or specificity nucleotide sequence.

8. the method for amplification in vitro purpose nucleotide sequence according to claim 11 is characterized in that at least 1 base pair of described intervening sequence.

9. the method for amplification in vitro purpose nucleotide sequence according to claim 1 is characterized in that described connection product can carry out pcr amplification again after the seizure elution system is carried out purifying.

10. method according to claim 9 is characterized in that described seizure elution system comprises:

1) vitamin H carries out mark to target nucleotide, and with the target nucleotide of streptavidin or avidin seizure mark, after extending connection and washing, wash-out is collected the connection product;

2) digoxin is carried out mark to target nucleotide, and with the target nucleotide of anti digoxin antibody seizure mark, after extending connection and washing, wash-out is collected the connection product;

3) complementary oligonucleotide that adheres to magnetic bead or latex bead.

11. the method for amplification in vitro purpose nucleotide sequence according to claim 1 is characterized in that described detection system comprises the chip hybridization detection system.

12. the method for amplification in vitro purpose nucleotide sequence according to claim 6 is characterized in that described detection system comprises:

1) detection system of PCR-based product length;

2) based on mass spectral detection system.

13. the multiple detection method of a plurality of purpose nucleotide sequences in the single sample of amplification, described method comprises:

(1) form polymerization-ligation mixture, comprising:

(i) target nucleic acid sequence;

(ii) oligonucleotide probe, the required oligonucleotide probe of each purpose nucleotide sequence amplification comprises the upstream oligonucleotide probe, its 3 ' end has at least a part of first section with first end of target nucleic acid sequence complementary basically, the downstream oligonucleotide probe, its 5 ' end has at least a part of second section with second end of target nucleic acid sequence complementary basically, in the intervening sequence of purpose nucleotide sequence between first section and second section, wherein terminal the and downstream oligonucleotide probe of upstream oligonucleotide probe 5 ' 3 ' is terminal respectively contains one section universal primer sequence respectively;

(iii) at least 4 kinds of different nucleotide bases;

(iv) polysaccharase and ligase enzyme;

(2) utilize universal primer, the upstream oligonucleotide probe extension sequence that amplification has connected and the product that is connected of downstream oligonucleotide probe produce a large amount of amplicons;

(3) detect described a plurality of amplicons, be present in the described sample to show described purpose nucleotide sequence.

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