CN101182514A - Isothermal PCR nucleic acid augmentative method based on dI modified primer and endonuclease V - Google Patents
Isothermal PCR nucleic acid augmentative method based on dI modified primer and endonuclease V Download PDFInfo
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Abstract
The invention relates to an isothermal PCR nucleic acid amplification method based on dI modified primer and endonucleaseV. The amplified target nucleic acid primer is completely matched with the template; 1 to 5 dI nucleic acids are on the 10 to 30 site at the 5' end; 10 to 30 nucleic acids are at the downstream of the dI nucleic acids. The reaction components of isothermal PCR comprise dI modified forward primer and reverse primer, target nucleic acid template, four kinds of deoxyribonucleoside triphosphates dATP, dTTP, dCTP and dGTP, DNA polymerase, endonucleaseV, single DNA chain binding protein and helicase. After the initial thermal denaturation and annealing hybridization, the mixture of isothermal PCR carries out the PCR reaction. During the amplification process, the circulation and regeneration of the primer-template compound is realized by endonucleaseV, which induces the DNA synthesis reaction; the single DNA chain binding protein and helicase can improve the efficiency of isothermal PCR. The amplification of the target nucleic acid of the invention does not rely on the thermal cycler; the invention has easy and convenient operation, which can be used for a large scale and high yield amplification of the target nucleic acid.
Description
Technical field
The present invention relates to a kind of new nucleic acid amplification method, relate in particular to a kind of isothermal PCR nucleic acid amplification method, can be used for the isothermal duplication target nucleic acid, belong to gene engineering technology field based on dI Mdification primer and endonuclease V.
Background technology
(Polymerase Chain Reaction PCR) is a kind of conventional Protocols in Molecular Biology that numerous application are arranged in the polymerase chain reaction.Conventional PCR mainly is made up of three-step reaction: the 1) thermally denature of target nucleic acid template molecule, 2) hybridization between the target nucleic acid template molecule of free primer and sex change annealing, the 3) synthesis reaction of DNA of the primer of thermostability archaeal dna polymerase catalyzed combination on the target nucleic acid template molecule.The three-step reaction recirculation of conventional PCR is carried out, and realizes the index multiple amplification of target nucleic acid.Conventional PCR needs expensive thermal cycler.Isothermal PCR is a kind of novel nucleic acids amplification technique, does not need thermal cycler.In isothermal PCR, primer and target nucleic acid template molecule are annealed through sex change, form the primer-template composite of complementary pairing, under steady temperature (generally being the optimal reactive temperature of archaeal dna polymerase) by archaeal dna polymerase catalytic dna building-up reactions continue carry out.Isothermal PCR need generate primer-template composite (effective substrate of synthesis reaction of DNA) constantly automatically in the DNA building-up process, so that synthesis reaction of DNA can continue to carry out.Because isothermal PCR does not rely on thermal cycler, can react in water-bath, the operating space is big, can enlarge reaction volume and flux easily.
Isothermal PCR at present commonly used mainly contains two kinds: based on the isothermal PCR of rolling-circle replication with based on isothermal PCR [Nucleic Acids Res., 2006, the 34:e98 of loop structure primer; Genome Res., 2001,11:1095-1099; Nucleic Acids Res., 2000,28:e63.].Wherein, be divided into catalytic isothermal PCR of phi 29 archaeal dna polymerases and the catalytic isothermal PCR of T7 archaeal dna polymerase based on the isothermal PCR of rolling-circle replication difference according to archaeal dna polymerase.In the catalytic isothermal PCR of phi 29 archaeal dna polymerases, forward primer is incorporated into cyclic single strand target nucleic acid template molecule, phi 29 archaeal dna polymerases are with the synthetic long single stranded DNA of form displacement of rolling-circle replication under 37 degree, reverse primer is incorporated into the long single stranded DNA of rolling-circle replication synthetic, the double-stranded DNA that composition length is different.Similar with phi 29 archaeal dna polymerases, the T7 archaeal dna polymerase is a template with the cyclic DNA also, utilizes forward primer and reverse primer, the different numerous double-stranded DNAs of composition length under 37 degree.Isothermal PCR based on loop structure primer is a template with the linear DNA, utilizes 4 primers, finally the double-stranded DNA of synthetic all lengths.
The defective of existing isothermal PCR technology mainly contains: 1) amplified production is the different double-stranded DNA mixture of length, need digest the dna fragmentation that forms the length homogeneous with restriction endonuclease; 2) phi 29 archaeal dna polymerases and the catalytic isothermal PCR of T7 archaeal dna polymerase can only amplification length relatively shorter ring-type target nucleic acid template molecule, and usually produce the non-specific amplification band; 3) though based on can the increase linear DNA molecule of high complexity of the isothermal PCR of loop structure primer, the limited length of its amplified fragments generally is lower than 250 bp; 4) isothermal PCR based on loop structure primer needs two pairs of primers, the amplified reaction complicated operation, and the failure of the synthesis reaction of DNA that any a pair of primer causes all can cause the failure of target nucleic acid amplification.
Summary of the invention
The objective of the invention is to deficiency at existing isothermal PCR technology, a kind of isothermal PCR nucleic acid amplification method based on dI Mdification primer and endonuclease V is provided, this isothermal PCR is easy and simple to handle, amplification efficiency, expanding fragment length and specificity improve greatly, can be used for the target nucleic acid fragment of isothermal duplication length homogeneous.
For realizing such purpose, the present invention realizes the regeneration of primer-template composite by endonuclease V and dI Mdification primer.In the present invention, the forward primer and the reverse primer of the target nucleic acid that is used to increase have following feature: whole piece primer and target nucleic acid template molecule match fully, and 1-5 dI Nucleotide is contained in 5 ' end 10-30 position, and the dI nucleotides downstream has 10-30 Nucleotide.The isothermal PCR reactive component comprises forward primer that dI modifies and reverse primer, target nucleic acid template molecule, dATP, dTTP, dCTP, dGTP totally 4 kinds of deoxyribonucleoside triphosphates, archaeal dna polymerase, endonuclease V, single-stranded DNA binding protein and helicases.The isothermal PCR mixture carries out PCR after annealing through initial thermally denature, hybridization under steady temperature.In amplification procedure, realize the cyclic regeneration of primer-template composite, cause synthesis reaction of DNA by endonuclease V; Single-stranded DNA binding protein and helicase can improve the target nucleic acid amplification efficiency of isothermal PCR.
The concrete steps of the inventive method are as follows:
1, the forward primer and the reverse primer of the synthetic target nucleic acid that is used to increase of design, the sequence signature of forward primer and reverse primer is: whole piece primer and target nucleic acid template molecule match fully, 1-5 dI Nucleotide is contained in 5 ' end 10-30 position, and the dI nucleotides downstream has 10-30 Nucleotide;
2, will be used to increase segmental forward primer of target nucleic acid and reverse primer, target nucleic acid template molecule and dATP, dTTP, dCTP, dGTP totally four kinds of deoxyribonucleoside triphosphates add the PCR damping fluids, be configured to not have the isothermal PCR mixture of protein ingredient; With archaeal dna polymerase (for example the big fragment of Klenow of the Bst archaeal dna polymerase of phi29 archaeal dna polymerase, T7 archaeal dna polymerase, the order-checking level Taq archaeal dna polymerase that does not have 5 ' 5 prime excision enzyme activity or 5 ' 5 prime excision enzyme activity disappearance etc.), endonuclease V, single-stranded DNA binding protein and helicase totally four kinds of protein, with 1-5 unit: 5-500 nanogram: 50-10000 nanogram: the mixed of 5-500 nanogram, according to four kinds of proteinic thermostabilitys, be made into the high temperature resistant egg white mixture of isothermal PCR normal temperature egg white mixture or isothermal PCR;
3, the isothermal PCR mixture that will not have protein ingredient is spent thermally denatures 5-10 minute 95, is cooled to then in the 25-37 degree scope and places 5 minutes, carries out the hybridization annealing of primer and target nucleic acid template molecule, forms primer-template composite; Add isothermal PCR normal temperature egg white mixture then, in 25-37 degree scope, carry out isothermal PCR, reaction times 1-24 hour;
Perhaps, the isothermal PCR mixture of no protein ingredient is blended directly in the high temperature resistant egg white mixture of isothermal PCR,, is cooled to then in the 42-72 degree scope and carries out isothermal PCR, reaction times 1-24 hour 95 degree thermally denatures 5-10 minute;
4) mixture behind the isothermal PCR being placed mass concentration is 1% sepharose, and electrophoresis is 20 minutes under the 200V condition, detects the target nucleic acid amplified production.
The present invention has compared obvious improvement with existing isothermal PCR with the thermal cycling round pcr.Major advantage is as follows: a certain steady temperature of (1) amplified reaction in 25-37 degree or 42-72 degree scope carried out, and do not need thermal cycler.(2) utilize endonuclease V and dI Mdification primer to realize the cyclic regeneration of primer-template composite, the synthesis reaction of DNA efficient of regenerated primer-template composite is suitable with thermal cycling PCR.(3) target nucleic acid increases with index amplification form simultaneously with linear, and amplification rate is higher than conventional PCR.(4) target nucleic acid amplified production specificity height though the dI Mdification primer may portion paired take place in the zone, many places of target nucleic acid template molecule, can produce PCR when having only primer and template molecule to match fully.(5) target nucleic acid expanding fragment length homogeneous, because it is conjuncted not form the repeated strings of amplified fragments in amplification procedure, so target nucleic acid expanding fragment length homogeneous.(6) helicase and single-stranded DNA binding protein make DNA resultant velocity and efficient improve greatly.(7) easy and simple to handle, result is stable, owing to do not need thermal cycler to generate primer-template composite, does not also just need to optimize a plurality of target nucleic acids needed general annealing temperature that increases simultaneously, amplification when greatly facilitating a plurality of target nucleic acid.(8) operation flux is big, does not need thermal cycler in the entire operation process, only needs this constant temperature heating device of water-bath, therefore can increase the amplified reaction number and the reaction volume of target nucleic acid easily, carries out the extensive high yield amplification of target nucleic acid.
The present invention can be used for the strand target nucleic acids such as cDNA of amplifying doulbe-chain target nucleic acid and RNA reverse transcription.Amplified reaction does not need the expensive thermal cycler primer-template composite of regenerating, and plant and instrument easy and simple to handle, required is simple, has enlarged the application places of target nucleic acid amplification greatly.Be mainly used in fields such as microorganism detection, gene clone, legal medical expert's evaluation.
Embodiment
By the following examples technical scheme of the present invention is described in further detail.Following examples do not constitute limitation of the invention.
Following examples utilize 5 ' the 19th at end to be the forward primer and the reverse primer of dI modified nucleotide, under the acting in conjunction of archaeal dna polymerase, endonuclease V, single-stranded DNA binding protein and helicase, finish the amplification of target nucleic acid.
The isothermal PCR amplification of the ampicillin resistance gene in the embodiment 1pUC18 plasmid
Target nucleic acid template molecule in the present embodiment is a plasmid pUC18, target nucleic acid to be amplified is the ampicillin resistance gene of plasmid pUC18, and four kinds of normal temperature protein that use are single-stranded DNA binding protein and the helicase of phi 29 archaeal dna polymerases, intestinal bacteria endonuclease V, T7 phage.Concrete implementation step is as follows:
The first step, design is synthetic be used to the to increase primer sequence of ampicillin resistance gene of plasmid pUC18.Article 2, primer is respectively pUC18-amp-F, pUC18-amp-R.Article 2, the primer base sequence is as follows:
pUC18-amp-F：5’aatattgaaa?aaggaagaxt?atgagtattc?aacatttccg?t
pUC18-amp-R：5’gagtaaactt?ggtctgacxg?ttaccaatgc?ttaatcagtg?a
Wherein, symbol X represents the dI modified nucleotide, the two ends complementary pairing of the ampicillin resistance gene of whole piece primer and pUC18 plasmid vector.Primer can oneself utilize dna synthesizer synthetic or synthetic by each DNA Synesis Company.
Second step, the preparation of isothermal PCR mixture.The isothermal PCR mixture is made of two portions: the isothermal PCR mixture of no protein ingredient and the isothermal PCR normal temperature egg white mixture of being made up of four kinds of albumen.The isothermal PCR mixture of no protein ingredient is formed (40 microlitre): forward primer and reverse primer, 1.0 μ M; The target nucleic acid template molecule, 5 nanogram pUC18 plasmids; DATP, dTTP, dCTP, dGTP, 200 μ M; 10 * phi, 29 dna polymerase reaction damping fluids, 4 microlitres; Adding deionized water to cumulative volume is 40 microlitres.Four kinds of isothermal PCR normal temperature egg white mixture compositions that albumen constitutes: phi 29 archaeal dna polymerases, 2.5 units; Intestinal bacteria endonuclease V, 50 nanograms; DNA is conjugated protein for the T7 phage single-chain, 500 nanograms; T7 phage helicase, 50 nanograms; 10 * phi, 29 dna polymerase reaction damping fluids, 1 microlitre; Adding deionized water to cumulative volume is 10 microlitres.
The 3rd step, the thermally denature of isothermal PCR mixture, annealing and isothermal PCR reaction.The isothermal PCR mixture of the no protein ingredient of 40 microlitres 95 degree thermally denatures 5-10 minute, is cooled to 37 degree insulations 5 minutes then, dI Mdification primer and pUC18 plasmid hybridization are matched, form initial primer-template composite; Add 10 microlitre isothermal PCR normal temperature egg white mixtures then, final isothermal PCR mixture (50 microlitre) was bathed 2 hours in 37 degree temperature, and the DNA that carries out isothermal PCR continues synthetic.
In the 4th step, acillin resistant gene amplification detects.It is 1% sepharose that mixture behind the isothermal PCR is placed mass concentration, and having added final concentration in the gel is the bromination second pyridine of 0.5 mcg/ml.Utilize the horizontal strip electrophoresis instrument, electrophoresis is 20 minutes under 200V voltage, observation acillin resistant gene amplification under UV-light or in the gel imaging system, and acillin resistant gene amplified production is the dna segment of 861 bp.
The principle of isothermal PCR amplification target nucleic acid: the first step, thermally denature-annealing form primer-template composite, archaeal dna polymerase (catalytic dna building-up reactions) generates a large amount of strand target nucleic acid molecules with the acting in conjunction of endonuclease V (cyclic regeneration of catalysis primer-template composite); In second step, strand target nucleic acid molecules that the first step generates and the pairing of free primer form new primer-template composite, cause the synthesis reaction of DNA of exponential form, generate a large amount of strand target nucleic acid molecules; The 3rd step, along with a large amount of minimizings of free primer, and the rolling up of synthetic strand target nucleic acid molecules, the strand target nucleic acid molecules is hybridized pairing each other and is formed the double-stranded DNA amplified production.A large amount of strand target nucleic acid molecules that single-stranded DNA binding protein generates in can the association reaction process make it be in the strand state; The DNA building-up process double center chain DNA that helicase promotion primer-template composite is initial unwinds; The acting in conjunction of single-stranded DNA binding protein and helicase can improve the target nucleic acid amplification efficiency of isothermal PCR.
The isothermal PCR of endonuclease IV gene amplification in the embodiment 2 chlamydia trachomatis gene groups
Target nucleic acid template molecule in the present embodiment is chlamydia trachomatis gene group DNA, target nucleic acid to be amplified is chlamydial endonuclease IV gene, four kinds of high temperature resistant protein that use are the Taq archaeal dna polymerase, endonuclease V, single-stranded DNA binding protein and the helicase of high temperature resistant microorganism T.thermophilus.Concrete implementation step is as follows:
The first step, design is synthetic be used to the to increase primer sequence of chlamydozoan endonuclease IV gene.Article 2, primer is respectively CpendoIV-F, CpendoIV-R.Article 2, the primer base sequence is as follows:
CpendoIV-F：5’agaaaagacc?ttgaaattxt?atgaaagtac?ttcctcctcc?c
CpendoIV-R：5’aaaagcactt?aaaaaactxc?ctaactatct?ctgttttttg?a
Wherein, symbol X represents the dI modified nucleotide, whole piece primer and chlamydial endonuclease IV gene two ends complementary pairing.Primer can oneself utilize dna synthesizer synthetic or synthetic by each DNA Synesis Company.
Second step, the preparation of isothermal PCR mixture.The two mixes the isothermal PCR mixture by the isothermal PCR mixture of no protein ingredient and the high temperature resistant egg white mixture of isothermal PCR.Final isothermal PCR mixture is formed (50 microlitre): forward primer and reverse primer, 1.0 μ M; The target nucleic acid template molecule, 50 nanogram chlamydia trachomatis gene group DNA; DATP, dTTP, dCTP, dGTP, 200 μ M; The Taq archaeal dna polymerase, 2.5 units; The endonuclease V of T.thermophilus, 50 nanograms; The single-stranded DNA binding protein of T.thermophilus, 500 nanograms; The helicase of T.thermophilus, 50 nanograms; 10 * Taq dna polymerase reaction damping fluid, 5 microlitres; Adding deionized water to cumulative volume is 50 microlitres.
The 3rd step, the thermally denature of isothermal PCR mixture, annealing and isothermal PCR reaction.The isothermal PCR mixture of 50 microlitres is spent thermally denatures 5-10 minute 95, be cooled to then about 60 degree, and continue temperature and bathed 2 hours in water-bath, the DNA that carries out isothermal PCR is lasting synthetic.
In the 4th step, chlamydozoan endonuclease IV gene amplification result detects.It is 1% sepharose that mixture behind the isothermal PCR is placed mass concentration, and having added final concentration in the gel is the bromination second pyridine of 0.5 mcg/ml.Utilize the horizontal strip electrophoresis instrument, electrophoresis is 20 minutes under 200V voltage, observation chlamydozoan endonuclease IV gene amplification result under UV-light or in the gel imaging system, and chlamydozoan endonuclease IV gene amplification product is the dna segment of 882bp.
The isothermal PCR amplification of embodiment 3 human alpha-defensin α 3mRNA
Template in the present embodiment is total cDNA of human total rna reverse transcription, target nucleic acid to be amplified is a human alpha-defensin α 3cDNA gene, and four kinds of normal temperature protein that use are phage t7 archaeal dna polymerase (combining escherichia coli thioredoxin), T7 single-stranded DNA binding protein, T7 helicase and intestinal bacteria endonuclease V.
Before utilizing isothermal PCR amplification human alpha-defensin α 3cDNA gene of the present invention, need to prepare the total cDNA of people by reverse transcription reaction in advance.Reverse transcription reaction is formed: contain human total rna 3 micrograms in the 100ul reverse transcription reaction mixture, 1 * reverse transcription damping fluid, the dATP of 0.5mM, dTTP, dCTP, dGTP, the oligo-dT Oligonucleolide primers of 2 μ M, the RNase A inhibitor of 10 units, 10 M-MLV of unit ThermoScript II.In 37 degree reactions 60 minutes.Column chromatography is removed the deoxyribonucleoside triphosphate that does not mix, and reclaims total cDNA, and is dissolved in 100ul water, measures total cDNA concentration.
The concrete implementation step of the inventive method is as follows:
The first step, design is synthetic be used to the to increase primer sequence of human alpha-defensin α 3 encoding genes.Article 2, primer is respectively DEFA3-F, DEFA3-R.Article 2, the primer base sequence is as follows:
DEFA3-F：5’gaggatctgt?gaccccagxc?atgaggaccc?tcgccatcct?t
DEFA3-R：5’atttttcttt?ttctgcaaxc?tcagcagcag?aatgcccaga?g
Wherein, symbol X represents the dI modified nucleotide, the cDNA two ends complementary pairing of whole piece primer and human alpha-defensin α 3 encoding genes.Primer can oneself utilize dna synthesizer synthetic or synthetic by each DNA Synesis Company.
Second step, the preparation of isothermal PCR reaction mixture.The isothermal PCR reaction mixture is made of two portions: the isothermal PCR mixture of no protein ingredient and isothermal PCR normal temperature egg white mixture.The isothermal PCR mixture of no protein ingredient is formed (40 microlitre): forward primer and reverse primer, 1.0 μ M; The target nucleic acid template molecule, the total cDNA of 50 nanogram people; DATP, dTTP, dCTP, dGTP, 200 μ M; 10 * T7 dna polymerase reaction damping fluid, 4 microlitres; Adding deionized water to cumulative volume is 40 microlitres.Isothermal PCR normal temperature egg white mixture is formed: T7 archaeal dna polymerase, 2.5 units; Intestinal bacteria endonuclease V, 50 nanograms; The T7 single-stranded DNA binding protein, 500 nanograms; The T7 helicase, 50 nanograms; 10 * T7 dna polymerase reaction damping fluid, 1 microlitre; Adding deionized water to cumulative volume is 10 microlitres.
The 3rd step, the thermally denature of isothermal PCR reaction mixture, annealing and isothermal PCR reaction.With the isothermal PCR mixture of the no protein ingredient of 40 microlitres 95 degree thermally denatures 5-10 minute, be cooled to 37 degree then and continue insulation 5 minutes, make the cDNA target nucleic acid template molecule hybridization pairing of dI Mdification primer and human alpha-defensin α 3, form initial primer-template composite; The isothermal PCR normal temperature egg white mixture that adds 10 microlitres was then bathed 2 hours in 37 degree temperature, and the DNA that carries out isothermal PCR continues synthetic.
In the 4th step, human alpha-defensin α 3 cDNA gene amplification results detect.It is 1% sepharose that mixture behind the isothermal PCR is placed mass concentration, and having added final concentration in the gel is the bromination second pyridine of 0.5 mcg/ml.Utilize the horizontal strip electrophoresis instrument, electrophoresis is 20 minutes under 200V voltage, observation human alpha-defensin α 3 cDNA gene amplification results under UV-light or in the gel imaging system, and human alpha-defensin α 3 cDNA gene amplification products are the dna segment of 285bp.
Claims (1)
1. isothermal PCR nucleic acid amplification method based on dI Mdification primer and endonuclease V is characterized in that may further comprise the steps:
1) forward and the reverse primer sequence of the synthetic target nucleic acid that is used to increase of design, the sequence signature of forward primer and reverse primer is: whole piece primer and target nucleic acid template molecule match fully, 1-5 dI Nucleotide is contained in 5 ' end 10-30 position, and the dI nucleotides downstream has 10-30 Nucleotide;
2) will be used to increase segmental forward primer of target nucleic acid and reverse primer, target nucleic acid template molecule and dATP, dTTP, dCTP, dGTP totally four kinds of deoxyribonucleoside triphosphates add the polymerase chain reaction damping fluids, be configured to not have the isothermal polymerase chain reaction mixture of protein ingredient; With archaeal dna polymerase, endonuclease V, single-stranded DNA binding protein and helicase totally four kinds of protein, with 1-5 unit: 5-500 nanogram: 50-10000 nanogram: the mixed of 5-500 nanogram, according to four kinds of proteinic thermostabilitys, be made into the high temperature resistant egg white mixture of isothermal polymerase chain reaction normal temperature egg white mixture or isothermal polymerase chain reaction;
3) the isothermal polymerase chain reaction mixture that will not have protein ingredient is spent thermally denatures 5-10 minute 95, is cooled to then in the 25-37 degree scope and places 5 minutes, carries out the hybridization annealing of primer and target nucleic acid template molecule, forms primer-template composite; Add isothermal polymerase chain reaction normal temperature egg white mixture then, in 25-37 degree scope, carry out the isothermal polymerase chain reaction, reaction times 1-24 hour;
Perhaps, the isothermal polymerase chain reaction mixture of no protein ingredient is blended directly in the high temperature resistant egg white mixture in isothermal polymerase chain reaction, spend thermally denatures 5-10 minute 95, be cooled to then in the 42-72 degree scope and carry out the isothermal polymerase chain reaction, reaction times 1-24 hour;
4) mixture behind the isothermal polymerase chain reaction being placed mass concentration is 1% sepharose, and electrophoresis is 20 minutes under the 200V condition, detects the target nucleic acid amplified production.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103472236A (en) * | 2013-09-11 | 2013-12-25 | 深圳先进技术研究院 | Method for detecting DNA (deoxyribonucleic acid) binding protein |
| CN104830698A (en) * | 2015-04-30 | 2015-08-12 | 广西壮族自治区农业科学院甘蔗研究所 | Pokkah boeng disease pathogen separating and identifying method |
| CN109266670A (en) * | 2018-10-21 | 2019-01-25 | 苏州博睐恒生物科技有限公司 | The method for carrying out gene cloning and point mutation using dI base |
| CN115807092A (en) * | 2022-11-25 | 2023-03-17 | 珠海圣美生物诊断技术有限公司 | Primer, probe and kit for detecting MET gene exon 14 skipping mutation |
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2007
- 2007-11-08 CN CNA2007100479533A patent/CN101182514A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103472236A (en) * | 2013-09-11 | 2013-12-25 | 深圳先进技术研究院 | Method for detecting DNA (deoxyribonucleic acid) binding protein |
| CN103472236B (en) * | 2013-09-11 | 2015-06-03 | 深圳先进技术研究院 | Method for detecting DNA (deoxyribonucleic acid) binding protein |
| CN104830698A (en) * | 2015-04-30 | 2015-08-12 | 广西壮族自治区农业科学院甘蔗研究所 | Pokkah boeng disease pathogen separating and identifying method |
| CN109266670A (en) * | 2018-10-21 | 2019-01-25 | 苏州博睐恒生物科技有限公司 | The method for carrying out gene cloning and point mutation using dI base |
| CN115807092A (en) * | 2022-11-25 | 2023-03-17 | 珠海圣美生物诊断技术有限公司 | Primer, probe and kit for detecting MET gene exon 14 skipping mutation |
| CN115807092B (en) * | 2022-11-25 | 2023-11-03 | 珠海圣美生物诊断技术有限公司 | Primer, probe and kit for detecting jump mutation of MET gene No. 14 exon |
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