CN103421777A - PCR-instrument-free method for fast and accurately amplifying lateral wing nucleotide sequence and application - Google Patents
PCR-instrument-free method for fast and accurately amplifying lateral wing nucleotide sequence and application Download PDFInfo
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Abstract
The invention provides a PCR-instrument-free method for fast and accurately amplifying a lateral wing nucleotide sequence. The method comprises the following steps of 1) a first round of reaction, wherein the first round of reaction is carried out through a DNA template, recombinase polymerase and a first round of primers; 2) a second round of reaction, wherein the product of the first round of reaction is used as a template, recombinase polymerase and a second round of primers are added, and the second round of reaction is carried out; 3) a third round of reaction, wherein the product of the second round of reaction is used as a template, recombinase polymerase and a third round of primers are added, and the third round of reaction is carried out. The primers and kits for the PCR-instrument-free method for fast and accurately amplifying the lateral wing nucleotide sequence are further provided. The invention further provides the application of the method, the primers and the kits in the amplification of a DNA sequence. The method is free of the PCR instrument, and can amplify the DNA lateral wing sequence simply, efficiently and accurately.
Description
Technical field
The present invention relates to the genetically engineered field, particularly relate to quick and precisely amplification method and the application of a kind of nucleotide sequence of the flank without the PCR instrument.
Background technology
The scientific research personnel has been developed several PCR method without setting up cDNA storehouse or screening and cloning, for obtaining the unknown nucleotide sequence be connected with known array.These PCR method comprise inverse PCR (IPCR), ligation-mediated PCR (LM-PCR), arbitrarily primed PCR (RP-PCR) etc.Although these methods have certain effect and updated, still be limited by that loaded down with trivial details enzyme is cut, aptamers connects (adaptor ligation) and purification step.And the random primer of RP-PCR often can produce unnecessary product because of non-specific annealing.Renaturation is afterwards controlled the specificity that primer (ACP) technology has improved the small pieces oligonucleotide, can overcome some shortcomings of appeal gene walking method, but this technology still relies on the amplification of PCR instrument, three-wheel PCR reaction time consumption reaches 4 and a half hours, and, during the given block primer be complementary at design and renaturation control primer, still to consider primer annealing.
Summary of the invention
For addressing the above problem, the purpose of this invention is to provide quick and precisely amplification method and the application of a kind of nucleotide sequence of the flank without the PCR instrument.
The present invention is combining recombinase polymeric enzymatic amplification technology (Recombinase Polymerase Amplification) of success first with containing hypoxanthic design of primers technology, and realize the efficiently and accurately amplification of DNA flanking sequence.
The present invention selects the recombinase polysaccharase of Britain TwistDX company, they have fast, the characteristics of isothermal, normal temperature amplification, it is a kind of amplification technique that is different from PCR fully, the nucleic acid primer of recombinase pairing DNA profiling wherein, polysaccharase synthesizes the new nucleic acid chain, can about 15 minutes, complete exponential amplification.
Exist following problem in traditional arbitrarily primed PCR process: random primer is too short, likely causes many wheel PCR products inconsistent, and random primer is long, likely increases less than product.For addressing this problem, we introduce poly deoxidation inosine poly (dI) in the random primer design process, can make like this more than random primer length reaches 20bp, effectively to reduce the unstable that cause because primer length is too short in many wheel amplifications; Do not affect the randomness of random primer when template is combined owing to introducing poly (dI), doing the used time with template DNA, poly in primer (dI) can form the structure (as shown in Figure 1) of an air bubble-shaped, it can promote poly (dI) front to be combined with template specificity by the sequence of 5 based compositions and suppress the combination of its back sequence and template, thus the template that only 5 bases of specific amplified and front end are combined.
The invention provides for the primer of amplification method quick and precisely of the flank nucleotide sequence without the PCR instrument, it is:
RPA-nest1c:tcacagaagtatgccaagcgaggiiiiicggtc(is as shown in SEQ ID No.1);
RPA-nest1a:tcacagaagtatgccaagcgaggiiiiiaggtc(is as shown in SEQ ID No.2);
RPA-nest1t:tcacagaagtatgccaagcgaggiiiiitggtc(is as shown in SEQ ID No.3);
RPA-nest1g:tcacagaagtatgccaagcgaggiiiiigggtc(is as shown in SEQ ID No.4);
RPA-nest2:tcacagaagtatgccaagcg(is as shown in SEQ ID No.5);
RPA-nest3:cagaagtatgccaagcgagg(is as shown in SEQ ID No.6).
A kind of nucleotide sequence of the flank without the PCR instrument provided by the invention is amplification method quick and precisely, comprises the steps:
1) first round reaction
Carry out first round reaction with DNA profiling, recombinase polysaccharase, first round primer;
Described first round primer is that RPA-nest1c(is as shown in SEQ ID No.1), RPA-nest1a(is as shown in SEQ ID No.2), RPA-nest1t(is as shown in SEQ ID No.3) and RPA-nest1g(as shown in SEQ ID No.4);
2) second take turns reaction
Take first round reaction product as template, add recombinase polysaccharase, second to take turns primer, carry out second and take turns reaction;
Described second to take turns primer be that RPA-nest2(is as shown in SEQ ID No.5);
3) third round reaction
Take and second take turns reaction product as template, add recombinase polysaccharase, third round primer to carry out the third round reaction;
Described third round primer is that RPA-nest3(is as shown in SEQ ID No.6).
Wherein, the recombinase polysaccharase that described recombinase polysaccharase is Britain TwistDX company, specifically TwistDX recombinase polysaccharase freezing dry powder (freeze-dried reaction).
Wherein, reaction system is specific as follows:
First round reaction system:
Wherein, reaction conditions is every 37-39 ℃ of heating 15-40 minute of reaction that take turns, and preferably heats 15 minutes.
Another object of the present invention is to provide for the test kit of amplification method quick and precisely of the flank nucleotide sequence without the PCR instrument, comprises following component:
Of the present invention for the test kit of amplification method quick and precisely of the flank nucleotide sequence without the PCR instrument, the test kit of 100 times of increasing as be designed to comprises following component:
Another object of the present invention is to provide the quick and precisely application of amplification method in the DNA sequence dna amplification of the described nucleotide sequence of the flank without the PCR instrument.
It is described for the application that quick and precisely primer of amplification method increases at DNA sequence dna of the flank nucleotide sequence without the PCR instrument that the present invention also provides.
It is described for the application that quick and precisely test kit of amplification method increases at DNA sequence dna of the flank nucleotide sequence without the PCR instrument that the present invention also provides.
Method of the present invention is invented for simple, efficient, accurate DNA amplification flanking sequence, be a kind ofly need not build storehouse, need not enzyme cut, need not fix, method that also need not complicated PCR, success first recombinase polymeric enzymatic amplification technology (Recombinase Polymerase Amplification) is combined and realizes that the efficiently and accurately of DNA flanking sequence increases with containing hypoxanthic design of primers technology, target product can obtain in 4 hours.Method of the present invention surmounts other existing methods, very reliable, only obtains target product, without non-specific product.Major advantage of the present invention is as follows:
(1) avoided because of sex change, annealing, extend equitemperature and control the wrong amplification failure caused.
(2) without the PCR instrument, easy and simple to handle, consuming time few, can in two hours, complete amplification and electrophoresis detection that the three-wheel nido reacts.
(3) effectively suppressed non-specific amplification because of design of primers (comprise in design of primers and add xanthoglobulin) cleverly, specific band nothing but after the three-wheel amplification.
The accompanying drawing explanation
Fig. 1 contains the random primer amplification schematic diagram that poly deoxidation inosine is arranged.
Wherein, 1 is 5 base sequences of 3' end, and 2 is poly deoxidation inosine, and 3 hold non-target afterbody sequence for 5'.
Fig. 2 is experiment flow and the electrophoresis photo of embodiment 1 with one section microsatellite sequence total length in method amplification Chinese chestnut of the present invention.
Wherein, Y1, Y2, Y3 are respectively the electrophoretic band of three-wheel product.
Embodiment
Following examples are used for the present invention is described, but are not used for limiting the scope of the invention.If do not specialize, the conventional means that technique means used is well known to those skilled in the art; Experimental technique used is ordinary method; Material used, reagent etc., all can obtain from commercial channels.
One section microsatellite sequence in Chinese chestnut:
GGCTGAAAACCAAGGCCGGAGTGGCATGTTCTTTAAAATGCTAT
CAGTAATCCCTCAGGTCAACTCTCTCTCTCTCTCTCTCTCTCGCT
CTCTCTCTCCATATATATATGTGTGTGTGTGTGTGTGTGCGCGCG
CGTGTGTGTGTGTGTG(is as shown in SEQ ID No.7)
This section microsatellite sequence total length amplification is very difficult: in this sequence, the tumor-necrosis factor glycoproteins of site tumor-necrosis factor glycoproteins reaches 82; There is multiple copied in the site partial sequence at genome; In site, there is reverse complementary sequence in the section of having sequence in genome.Inverse PCR (IPCR) with forefathers' invention, ligation-mediated PCR (LM-PCR), arbitrarily primed PCRs (RP-PCR) etc. all increase and arrive the purpose fragment, although control primer (ACP) the technology purpose fragment that increased by renaturation, but in a couple of days consuming time, and get into trouble in the process of design of primers, the careless slightly the failure of an experiment that causes of design of primers.
The concrete reaction method of the present embodiment is as follows:
1 according to known array design primer
The first round:
Random primer (synthetic by invitrogen company):
RPA-nest1c:tcacagaagtatgccaagcgaggiiiiicggtc(is as shown in SEQ ID No.1)
RPA-nest1a:tcacagaagtatgccaagcgaggiiiiiaggtc(is as shown in SEQ ID No.2)
RPA-nest1t:tcacagaagtatgccaagcgaggiiiiitggtc(is as shown in SEQ ID No.3)
RPA-nest1g:tcacagaagtatgccaagcgaggiiiiigggtc(is as shown in SEQ ID No.4)
Immobilized primer on known array:
F1:TCAGTAATCCCTCAGGTCAA(is as shown in SEQ ID No.8, synthetic by invitrogen company)
Second takes turns:
RPA-nest2:TCACAGAAGTATGCCAAGCG(is as shown in SEQ ID No.5, synthetic by invitrogen company)
F2:CAGTAATCCCTCAGGTCAAC(is as shown in SEQ ID No.9, synthetic by invitrogen company)
Third round:
RPA-nest3:CAGAAGTATGCCAAGCGAGG(is as shown in SEQ ID No.6, synthetic by invitrogen company)
F3:GTAATCCCTCAGGTCAACTC(is as shown in SEQ ID No.10, synthetic by invitrogen company)
2 nido amplification technique reaction systems:
First round reaction system: RPA-nest1c(is as shown in SEQ ID No.1) 0.6uL, RPA-nest1a(be as shown in SEQ ID No.2) 0.6uL, RPA-nest1t(be as shown in SEQ ID No.3) 0.6uL, RPA-nest1g(be as shown in SEQ ID No.4) immobilized primer F12.4uL, TwistDx Rehydration Buffer29.5uL, DNA profiling (10-1000ng) on 0.6uL, known array add ddH
2O 13.2uL altogether mixes, and adds TwistDX recombinase polysaccharase freezing dry powder 1pellet(1 pipe) mix, then add magnesium acetate (MgAc) aqueous solution that 2.5uL concentration is 280mmol/L to mix, obtain first round reaction system;
Second takes turns reaction system: RPA-nest2(as shown in SEQ ID No.5) immobilized primer F22.4uL, TwistDx Rehydration Buffer29.5uL, first round product 5uL, ddH on 2.4uL, known array
2O8.2uL mixes, and adds TwistDX recombinase polysaccharase freezing dry powder 1pellet and mixes, then add the MgAc aqueous solution that 2.5uL concentration is 280mmol/L to mix, and obtains second and takes turns reaction system;
Third round reaction system: RPA-nest3(is as shown in SEQ ID No.6) immobilized primer F32.4uL, TwistDx Rehydration Buffer29.5uL, second on 2.4uL, known array take turns product 5uL, ddH
2O8.2uL mixes, and adds TwistDX recombinase polysaccharase freezing dry powder 1pellet and mixes, then add the MgAc aqueous solution that 2.5uL concentration is 280mmol/L to mix, and obtains the third round reaction system.
Reaction conditions: in gold and silver apricot H2O3-100C dry type constant-temperature metal bath, heat, every take turns 37～39 ℃ 15 minutes.
3 electrophoresis detection
Get the final amplified production of 5uL point sample electrophoresis (adopting the Baygene horizontal cataphoresis apparatus) in agarose.The electrophoresis detection of three-wheel reaction and purifying and common PCR reaction are as good as, can be not purified with regard to electrophoresis detection, and the point sample sky is brighter but do not affect detected result.
4 purified products
With common PCR product purification test kit (as MinElute Reaction Cleanup Kit), get final product.
Fig. 2 is shown in by experiment flow and electrophoresis photo, and sequencing result is with SEQ ID No.7, and the method for the present invention target sequence total length that successfully increased is described.
Result shows, the recombinase polysaccharase flanking sequence amplification kit of we oneself exploitation can in two hours, complete the reaction of three-wheel nido and without the PCR instrument without groping complicated PCR response procedures, have time-saving and efficiency, design of primers requires low, product specificity advantages of higher.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements, all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (10)
1. for the primer of amplification method quick and precisely of the flank nucleotide sequence without the PCR instrument, it is:
RPA-nest1c:tcacagaagtatgccaagcgaggiiiiicggtc(is as shown in SEQ ID No.1);
RPA-nest1a:tcacagaagtatgccaagcgaggiiiiiaggtc(is as shown in SEQ ID No.2);
RPA-nest1t:tcacagaagtatgccaagcgaggiiiiitggtc(is as shown in SEQ ID No.3);
RPA-nest1g:tcacagaagtatgccaagcgaggiiiiigggtc(is as shown in SEQ ID No.4);
RPA-nest2:tcacagaagtatgccaagcg(is as shown in SEQ ID No.5);
RPA-nest3:cagaagtatgccaagcgagg(is as shown in SEQ ID No.6).
2. the nucleotide sequence of the flank without PCR instrument amplification method quick and precisely, is characterized in that, comprises the steps:
1) first round reaction
Carry out first round reaction with DNA profiling, recombinase polysaccharase, first round primer;
Described first round primer is that RPA-nest1c(is as shown in SEQ ID No.1), RPA-nest1a(is as shown in SEQ ID No.2), RPA-nest1t(is as shown in SEQ ID No.3) and RPA-nest1g(as shown in SEQ ID No.4);
2) second take turns reaction
Take first round reaction product as template, add recombinase polysaccharase, second to take turns primer, carry out second and take turns reaction;
Described second to take turns primer be that RPA-nest2(is as shown in SEQ ID No.5);
3) third round reaction
Take and second take turns reaction product as template, add recombinase polysaccharase, third round primer to carry out the third round reaction;
Described third round primer is that RPA-nest3(is as shown in SEQ ID No.6).
3. method according to claim 2, is characterized in that, the recombinase polysaccharase that described recombinase polysaccharase is Britain TwistDX company.
4. method according to claim 3, is characterized in that, described recombinase polysaccharase is TwistDX recombinase polysaccharase freezing dry powder.
5. method according to claim 2, is characterized in that, reaction system is specific as follows:
First round reaction system:
6. method according to claim 2, is characterized in that, reaction conditions is every 37-39 ℃ of heating 15-40 minute of reaction that take turns.
7. for the test kit of amplification method quick and precisely of the flank nucleotide sequence without the PCR instrument, comprise following component:
8. the application of the described method of claim 2-6 any one in the DNA sequence dna amplification.
9. the application of primer claimed in claim 1 in the DNA sequence dna amplification.
10. the application of test kit claimed in claim 7 in the DNA sequence dna amplification.
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| CN103881989A (en) * | 2014-03-06 | 2014-06-25 | 中国农业科学院生物技术研究所 | Novel DNA polymerase with continuous synthesis capability and salt tolerance |
| CN111621597A (en) * | 2020-05-09 | 2020-09-04 | 清华大学 | Virus recombinase-polymerase amplification detection method |
| CN112111602A (en) * | 2020-08-13 | 2020-12-22 | 安徽微分基因科技有限公司 | Kit and method for detecting COVID-19 virus by combination of nested isothermal amplification and gene editing |
| CN112501262A (en) * | 2020-12-15 | 2021-03-16 | 苏州点晶生物科技有限公司 | Nucleic acid amplification detection method and reagent based on dual multi-enzyme mediation |
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| WO2021226754A1 (en) * | 2020-05-09 | 2021-11-18 | 杭州梓晶生物有限公司 | Method for detecting viral recombinase polymerase amplification |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103881989A (en) * | 2014-03-06 | 2014-06-25 | 中国农业科学院生物技术研究所 | Novel DNA polymerase with continuous synthesis capability and salt tolerance |
| CN103881989B (en) * | 2014-03-06 | 2016-11-02 | 中国农业科学院生物技术研究所 | A kind of archaeal dna polymerase with processivity and salt tolerance level |
| US10047349B2 (en) | 2014-03-06 | 2018-08-14 | Hangzhou Zhongce Bio-Sci&Tech. Co., Ltd. | DNA polymerase possessing continuous catalytic capacity and salt tolerance |
| US10301606B2 (en) | 2014-03-06 | 2019-05-28 | Hangzhou Zhongee Bio-Sci & Tech. Co., Ltd. | DNA polymerase possessing continuous catalytic capacity and salt tolerance |
| CN111621597A (en) * | 2020-05-09 | 2020-09-04 | 清华大学 | Virus recombinase-polymerase amplification detection method |
| CN112111602A (en) * | 2020-08-13 | 2020-12-22 | 安徽微分基因科技有限公司 | Kit and method for detecting COVID-19 virus by combination of nested isothermal amplification and gene editing |
| CN112501262A (en) * | 2020-12-15 | 2021-03-16 | 苏州点晶生物科技有限公司 | Nucleic acid amplification detection method and reagent based on dual multi-enzyme mediation |
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