CN101705292B - Exonuclease III/I protection analysis based fluorescent biosensor method for detecting single nucleotide polymorphism - Google Patents
Exonuclease III/I protection analysis based fluorescent biosensor method for detecting single nucleotide polymorphism Download PDFInfo
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- CN101705292B CN101705292B CN2009102270235A CN200910227023A CN101705292B CN 101705292 B CN101705292 B CN 101705292B CN 2009102270235 A CN2009102270235 A CN 2009102270235A CN 200910227023 A CN200910227023 A CN 200910227023A CN 101705292 B CN101705292 B CN 101705292B
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- exonuclease iii
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Abstract
The invention discloses an exonuclease protection analysis based fluorescent biosensor method for detecting single nucleotide polymorphism, which comprises allele-specific extension of micromolecule modified single nucleotide, interaction between an oligonucleotide DNA strand primer with micromolecules after extension and binding protein, protection analysis of the exonuclease III/I and fluorescent quantitative detection of a hairpin type oligonucleotide DNA probe primer subjected to end protection based on double-strand indicating dye. The method adopts allele-specific extension of the primer to combine the micromolecule modified single nucleotide at 3' end of a specific primer and then combine the specificity of the binding protein of the micromolecule to protect the primer from being degraded by the exonuclease III/I, and detects single nucleotide polymorphism genotyping through fluorescence of the complex of the protected oligonucleotide DNA primer and double-strand inserting dye. The method features simple and convenient operation, economy, fast speed, sensitivity and strong specificity and is expected to provide a universal technology platform for screening and prenatal diagnosis of the population suffering from gene mutation related genetic diseases.
Description
Technical field
The invention belongs to a kind of bio-sensing method that detects SNP; Comprise small numerator modified single deoxynucleotide equipotential specificity elongation technology, the fluorescent quantitation that has the protection analysis of micromolecular oligonucleotide DNA strand primer and protein-bonded interaction and exonuclease III/I after the extension and carry out the duplex structure oligonucleotide DNA chain of terminal protection based on double-stranded indicating dye detects.
Background technology
Human many genetic diseasess such as α and beta Thalassemia disease etc. all are because single gene mutation produces.These genetic diseasess are one of topmost inborn defects of China, have a strong impact on the quality of the people of China, bring heavy economical load for society and family, therefore, need set up a kind of quick, sensitive, specific detection technique.The tradition detection in Gene Mutation is mainly by means of electrophoresis, mass spectrum or method for gene chip, and these methods need expensive precision instrument, and schedule of operation is complicated, and technological epitaxy is poor, and analytical cycle is long, carries out large-scale crowd examination and antenatal diagnosis so be inappropriate for.Present international main trend all concentrates on the dna modification enzyme and is the basis; The fidelity, safety and the sensitivity that utilize enzymic catalytic reaction to improve the transgenation authentication method develop easy and simple to handle, fast and reliable, low cost, high-throughout gene mutation analysis means.For example; Extend analysis etc. based on the equipotential specificity extension of archaeal dna polymerase or TaqMan probe analysis, the mononucleotide of circumscribed foundation; Connect the oligonucleotide of setting up based on the equipotential specificity of dna ligase and connect analysiss, ligase enzyme link analysis etc., based on the Invader analysis of DNA restriction endonuclease structure identification etc.But these technology need to adopt special fluorescence labeling probe (like double-tagging) usually, and analysis cost is higher.
Summary of the invention
The technical problem that the present invention will solve is; Deficiency to the prior art existence; A kind of biological method for sensing based on exonuclease III/I protection analyzing and testing SNP has been proposed; This method does not need special fluorescent mark, can be used for accurate, the rapid detection of SNP.
Technical scheme of the present invention is to protect the biological method for sensing of analyzing and testing SNP to comprise based on exonuclease III/I:
(1) has the protection analysis of micromolecular oligonucleotide DNA strand primer and protein-bonded interaction and exonuclease III/I after small numerator modified single deoxynucleotide equipotential specificity is extended and extended
(2) fluorescent quantitation that carries out the duplex structure oligonucleotide DNA chain of terminal protection based on double-stranded indicating dye detects.
Below the present invention made further specify.
Among the present invention, the hair clip type probe primer and the binding protein interactions that have extended the single deoxynucleotide of small molecules mark make it does not adopted the equipotential specificity of biotin labeled single deoxynucleotide to extend and exonuclease III/I protection by the detection of exonuclease III/I degraded and protected primer.
Further, said biological method for sensing based on exonuclease III/I protection analyzing and testing SNP adopts following steps to realize:
Four kinds of deoxynucleotides getting hair clip type probe primer and small molecules mark are in the PCR pipe; Under the situation of target compound to be measured and archaeal dna polymerase existence, carry out single base equipotential specificity polymerization extension; Add again small molecules conjugated protein with PCR pipe in, add exonuclease III/I reaction then.
The detection by quantitative of the hair clip type probe primer DNA chain of said terminal protection can adopt the double-stranded fluorescence intercalative dye indicator method of standard to detect.
Among the present invention, saidly realize by following step based on exonuclease III/I protection analyzing and testing SNP:
The equipotential specificity of the single deoxynucleotide of small molecules mark is extended: the TV of base extension is 25 μ L, in four different PCR pipes, carries out, and wherein every pipe comprises object chain to be detected; Four kinds of monodeoxyribonucleotides of Biotin-ddNTP of the detection probes 50pmol of 5pmol a kind of, 2unit Taq archaeal dna polymerase, 1 * dna polymerase buffer solution; Carry out 94 ℃ of sex change 3min, 94 ℃ of sex change 30s in the PCR pipe that is reflected at 200 μ L; 62 ℃ of annealing 30s; 68 ℃ are extended 10s, and totally 20 circulations rest on 4 ℃ at last.Product after extending stops extension at 80 ℃ of heat inactivation 30min.
Exonuclease III/I protection: get in the solution of streptavidin behind above-mentioned single base extension that 1.5 μ L concentration are 1mg/mL 37 ℃ of isothermal reactions 0.5 hour; Add the exonuclease I of 80U and the exonuclease III of 40U again, place 37 ℃ of isothermal reactions after 0,5 hour, be warmed up to 80 ℃ of reaction 10min, put out a fire to stop endonuclease reaction.
Notice that above reaction conditions is an optimal conditions, said liquor capacity all can change at double and not change optimal result.
Among the present invention; The detection by quantitative of said biological method for sensing based on exonuclease III/I protection analyzing and testing SNP can adopt the double-stranded fluorescent indicator method of standard to carry out, below be with double-stranded fluorescence intercalative dye SYBR-GREEN 1 indicator method to protect based on exonuclease III/I the analyzing and testing SNP the detection by quantitative step:
In the reaction soln after the said exonuclease III/I effect; Add 5 μ l SYBR-GREEN 1 (a kind of double-stranded fluorescence intercalative dye; Dilution in 1: 30000); Being diluted to 100 μ l with the exonuclease reaction buffer, putting into XRF, is that excitation wavelength, 520nm are that emission wavelength is surveyed its fluorescence letter intensity with 480nm.
The present invention proposes a kind of analysing and detecting method based on exonuclease III/I protection analyzing and testing SNP; This method need not fluorescent mark; Only need design the oligonucleotide probe primer of a hair clip type; Situation about existing at object chain extends below the deoxynucleotide of a small molecules mark, and small molecules promptly capable of using is not degraded by excision enzyme III/I with this primer of its binding protein interactions protection SNP is not carried out gene type; Its operation is fast and convenient, sensitive special, and amount of samples is few, easy and simple to handle, cost is lower, the rapid automatized realization of ability, and being expected provides a general technology platform for crowd's examination, antenatal diagnosis.
Embodiment:
Embodiment 1: protection is analyzed and is used for the poor 654 site mutation detection in β ground based on exonuclease III/I
1) the equipotential specificity of biotin labeled single deoxynucleotide is extended: the TV of base extension is 25L; In four different PCR pipes, carry out; Wherein comprise object chain to be detected in each pipe; The hair clip type oligonucleotide probe primer of 5pmol, primer sequence are GAA TTC CAA GCG CGA AG TTTT CTT CGC GCT TGG AAT TC TTTTTTCAC CAT TCT AAA GAA TAA CAG TGA TAA TTT CTG GGT TAA GG, a kind of base of four kinds of Biotin-dNTP (Invitrogen) of 50pmol; 2unit Taq archaeal dna polymerase (NEB); 1 * Tap archaeal dna polymerase standard buffer solution (10mM Tris-HCL (pH 8.6), 50mM KCL, 1.5mM MgCL
2), be reflected in the PCR pipe of 200 μ L and carry out, 94 ℃ of sex change 3min, 94 ℃ of sex change 30s, 62 ℃ of annealing 30s, 68 ℃ are extended 10s, carry out 20 circulations altogether, rest on 4 ℃ at last.Product after extending stops extension at 80 ℃ of heat inactivation 10min.
2) exonuclease III/I protection: get in the solution of streptavidin behind above-mentioned single base extension that 3 μ L concentration are 1mg/mL 37 ℃ of isothermal reactions 0.5 hour; Add the exonuclease I (NEB) of 80U and the exonuclease III (NEB) of 40U again, place 37 ℃ of isothermal reactions after 0,5 hour, be warmed up to 80 ℃ of reaction 10min, put out a fire to stop endonuclease reaction.
The detection of SNP; Add 5 μ l SYBR-GREEN 1 (a kind of double-stranded fluorescence intercalative dye; Dilution in 1: 30000); Cut end product to 100 μ l with exonuclease III reaction buffer (10mM Bis Tris Propane-HCL, 10mM MgCL2,1mMDTT (pH 7.0)) dilution enzyme; Putting into XRF, is that excitation wavelength, 520nm are that emission wavelength is surveyed its fluorescence intensity with 480nm.
According to above-mentioned steps 1), 2) standardized solution (concentration is from 100pM to 100nM) of the object chain (synthetic) of 8 four kinds of different genotype is detected; Write down the fluorescence intensity of each standardized solution; The fluorescence intensity contrast of the fluorescence intensity of unknown sample and the liquid of standard, thereby the genotype of definite unknown sample.
Embodiment 2: the sudden change detection that is used for K-ras gene the 12nd codon is analyzed in protection based on exonuclease III/I
1) the equipotential specificity of biotin labeled single deoxynucleotide is extended: the TV of base extension is 25L; In four different PCR pipes, carry out, wherein comprise object chain to be detected, the hair clip type oligonucleotide probe primer of 5pmol in each pipe; Primer sequence is (GAA TTC CAA GCG CGA AG TTTT CTT CGC GCT TGG AAT TCTTTTTTGAATATAAACTTGTGGTAGTTGGAGCTG); A kind of base of four kinds of Biotin-dNTP (Invitrogen) of 50pmol, 2unit Taq archaeal dna polymerase (NEB), 1 * Tap archaeal dna polymerase standard buffer solution (10mMTris-HCL (pH 8.6); 50mM KCL, 1.5mM MgCL
2), be reflected in the PCR pipe of 200 μ L and carry out, 94 ℃ of sex change 3min, 94 ℃ of sex change 30s, 65 ℃ of annealing 30s, 72 ℃ are extended 10s, carry out 20 circulations altogether, rest on 4 ℃ at last.Product after extending stops extension at 80 ℃ of heat inactivation 15min.
Exonuclease III/I protection: get in the solution of streptavidin behind above-mentioned single base extension that 3 μ L concentration are 1mg/mL 37 ℃ of isothermal reactions 0.5 hour; Add the exonuclease I (NEB) of 80U and the exonuclease III (NEB) of 40U again, place 37 ℃ of isothermal reactions after 0,5 hour, be warmed up to 80 ℃ of reaction 10min, put out a fire to stop endonuclease reaction.
2) detection of SNP; Add 5 μ l SYBR-GREEN 1 (a kind of double-stranded fluorescence intercalative dye; Dilution in 1: 30000); Cut end product to 100 μ l with exonuclease III reaction buffer (10mM Bis Tris Propane-HCL, 10mM MgCL2,1mMDTT (pH 7.0)) dilution enzyme; Putting into XRF, is that excitation wavelength, 520nm are that emission wavelength is surveyed its fluorescence intensity with 480nm.
According to above-mentioned steps 1), 2) standardized solution (concentration is from 100pM to 100nM) of the object chain (synthetic) of 8 four kinds of different genotype is detected; Write down the fluorescence intensity of each standardized solution; The fluorescence intensity contrast of the fluorescence intensity of unknown sample and the liquid of standard, thereby the genotype of definite unknown sample.
Claims (3)
1. biological method for sensing based on exonuclease III/I protection analyzing and testing SNP is characterized in that this method comprises:
(1) there is protein-bonded small numerator modified single deoxynucleotide to carry out degrading with exonuclease III/I after the equipotential specificity extends to the end and small molecules binding protein interactions of hair clip type probe primer with PCR;
(2) fluorescent quantitation that carries out the duplex structure oligonucleotide DNA chain of terminal protection based on double-stranded indicating dye detects;
This method is got rid of the purpose of medical diagnosis on disease.
2. according to the said biological method for sensing of claim 1 based on exonuclease III/I protection analyzing and testing SNP; It is characterized in that the hair clip type probe primer and the small molecules binding protein interactions that have extended the single deoxynucleotide that protein-bonded small molecules mark is arranged make it does not adopted the equipotential specificity of biotin labeled single deoxynucleotide to extend and exonuclease III/I protection by the detection of exonuclease III/I degraded and protected primer.
3. according to claim 1 or 2 said biological method for sensing, it is characterized in that it adopts following steps to realize based on exonuclease III/I protection analyzing and testing SNP:
Four kinds of deoxynucleotides getting hair clip type probe primer and small molecules mark are in the PCR pipe; Under the situation of target compound to be measured and archaeal dna polymerase existence, carry out single base equipotential specificity polymerization extension; Add again small molecules conjugated protein with PCR pipe in, add exonuclease III/I reaction then;
The detection by quantitative of the hair clip type probe primer DNA chain of said terminal protection adopts the double-stranded fluorescence intercalative dye indicator method of standard to detect.
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| CN102586462A (en) * | 2012-03-20 | 2012-07-18 | 湖南大学 | Multi-component chromatography single nucleotide polymorphism detection method for biologically coding based on general PCR amplification |
| CN103215366A (en) * | 2013-04-25 | 2013-07-24 | 中国科学院广州生物医药与健康研究院 | Detection method for multi-genotyping based on isothermal signal amplification of nuclease and hairpin DNA (deoxyribonucleic acid) probe |
| CN103589797A (en) * | 2013-11-12 | 2014-02-19 | 中国农业科学院蔬菜花卉研究所 | SNP (single nucleotide polymorphism) genotyping method and application thereof |
| TWI617667B (en) * | 2017-03-03 | 2018-03-11 | 長庚大學 | Detection method and detection kit for nucleic acid molecules |
| CN109652499B (en) * | 2017-10-12 | 2022-06-03 | 深圳华大智造科技股份有限公司 | Method and kit for rapidly detecting 3'-5' exoactivity or mismatch of DNA polymerase |
| CN113151405B (en) * | 2021-05-28 | 2022-09-09 | 生捷科技(杭州)有限公司 | SNP typing detection method |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101526466A (en) * | 2009-04-17 | 2009-09-09 | 湖南大学 | Fluorescence biological sensing technology based on interaction between terminal-protected analyzed detected small molecules of exonuclease III and combined protein |
| CN101526467A (en) * | 2009-04-17 | 2009-09-09 | 湖南大学 | Optical biological sensing technology based on interaction between terminal-protected analyzed detected small molecules of single-chain exonuclease I and combined protein |
| CN101533027A (en) * | 2009-04-17 | 2009-09-16 | 湖南大学 | Biosensing technology for detecting interaction of small molecules and binding protein based on protection and analysis of terminals of single-stranded exonuclease |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101526466A (en) * | 2009-04-17 | 2009-09-09 | 湖南大学 | Fluorescence biological sensing technology based on interaction between terminal-protected analyzed detected small molecules of exonuclease III and combined protein |
| CN101526467A (en) * | 2009-04-17 | 2009-09-09 | 湖南大学 | Optical biological sensing technology based on interaction between terminal-protected analyzed detected small molecules of single-chain exonuclease I and combined protein |
| CN101533027A (en) * | 2009-04-17 | 2009-09-16 | 湖南大学 | Biosensing technology for detecting interaction of small molecules and binding protein based on protection and analysis of terminals of single-stranded exonuclease |
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