CN105087517A - Recombinase complex and in-vitro homologous recombination seamless cloning method - Google Patents
Recombinase complex and in-vitro homologous recombination seamless cloning method Download PDFInfo
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Abstract
The invention relates to a recombinase complex which is derived from escherichia coli Rec homologous recombinase and comprises RecE, RecT and Gamma proteins. The invention also provides an in-vitro homologous recombination seamless cloning method, and the method only needs to mix a PCR (polymerase chain reaction) product having vector terminal sequences at two ends with a linearization cloning vector in a certain proportion and efficiently and directionally clone target DNA to any site of any vector under the catalysis of the recombinase complex, wherein the cloning positive rate can be not less than 95%. The method provided by the invention is particularly suitable for cloning large DNA fragments, can be used for overcoming the defects of complicated operation, high time consumption, high failure rate and the like of a conventional cloning method, can provide a quick, convenient and efficient cloning method for DNA in-vitro recombination, and lays an important foundation in the aspects of cytological basic research, industrial and agricultural production and medical care.
Description
Technical field
What the present invention relates to is DNA recombinant technology in molecular biology, and what be specifically related to is a kind of cloned nucleic acid molecule method of external homologous recombination and a kind of recombinase mixture.
Background technology
Modern biology research will depend on recombinant DNA technology to a great extent.The DNA Reorganization occurred in cell, is called In vivo recombination.Due to the development of new technology, can carry out DNA restructuring by manual operation in extracellular now, be called vitro recombination.
The clone obtaining gene by the restructuring of DNA is the basis of the structure of research gene, function and evolution.Gene clone technology, the vegetative propagation etc. of the molecule clone technology that is otherwise known as, recombinant DNA technology, genetically engineered, genetic manipulation or gene.In the process of whole gene clone, reconfiguring of DNA is a very crucial step, and it directly decides the success or not of whole clone.As time goes on, going deep into of research, increasing DNA recombination method is applied in the middle of production and scientific research.
Classical clone side is cut-connected to traditional enzyme
method is producedbeing born in early 1970s, is the gene clone method applied the earliest, is more common and conventional method, uses till today always.But in the experimentation of reality, the step of this method seems comparatively loaded down with trivial details time-consuming, need to select different restriction endonucleases to use according to cloned gene sequence, cost is higher.And part is rich in the suitable restriction enzyme site of gene clone Chang Yinwu of special palindromic sequence and failure.
Therefore to cut method of attachment significant to substitute traditional enzyme to develop a kind of efficient vitro recombination clone technology fast easy and simple to handle.
Summary of the invention
For achieving the above object, the invention provides a kind of recombinase mixture, this recombinase mixture derives from intestinal bacteria Rec homologous recombination enzyme, comprises RecE, RecT and Gamma albumen, wherein the NCBIGeneID:8183641 of Gamma albumen.
Further, the content ratio of RecE, RecT and Gamma albumen three kinds of recombinases is 1:1:2.
Present invention also offers the method for the seamless clone of a kind of external homologous recombination, the method comprises the following steps:
Step one: preparation linearizing cloning vector;
Step 2: prepare Insert Fragment PCR primer, by holding the terminal homologous sequence introducing this linearizing cloning vector at primer 5 ', make 5 ' of this Insert Fragment PCR primer end and 3 ' end respectively with the on all four sequence corresponding with this linearizing cloning vector two ends;
Step 3: the Insert Fragment PCR primer obtained in the linearizing cloning vector obtained in step one and step 2 is carried out recombining reaction under the effect of this recombinase mixture;
Step 4: the recombinant products obtained in step 3 is carried out cell transformation, obtains transformant;
Step 5: the transformant obtained in step 4 is cultivated, and carry out clone identification, obtain through clone identification be positive recombinant DNA molecules.
Further, in step one, this linearizing cloning vector is obtained by digestion with restriction enzyme or Inverse PCR amplification.
Further, digestion with restriction enzyme is single endonuclease digestion or double digestion.
Further, when adopting the method for Inverse PCR amplification, exo+ polymerase is used to increase to pre-linearizing cloning vector.
Further, in step 2, the design of primer is as follows:
Forward amplimer design:
5 '-upstream vector terminal homologous sequence+gene specific forward extension increasing sequence-3 ';
Reverse amplimer design:
The reverse extension increasing sequence of 3 '-gene specific+downstream vector terminal homologous sequence-5 '.
Further, in primer, the length of homologous sequence is 15bp ~ 20bp.Bp means base pair, for representing the length of DNA molecular.
Further, in step 3, when Insert Fragment PCR primer is more than or equal to 5kb, linearizing cloning vector and Insert Fragment PCR primer mol ratio are 1:10.Kb means kilobase pair, for representing the length of DNA molecular.
Further, in step 3, when Insert Fragment PCR primer is less than or equal to 2kb, linearizing cloning vector and Insert Fragment PCR primer mol ratio are 1:2.
According to above-mentioned steps, the plasmid identification extracted after the positive bacterium colony of PCR is cultivated, in proved invention, target dna is under the catalysis of this recombinase mixture, in vitro can simple and direct directed cloning efficiently in required carrier.What is called is simple and direct efficiently to be referred under the catalysis of recombinase mixture, only needs reaction can transform for 30 minutes, completes directed cloning, and clone's positive rate can reach more than 95%.According to lot of documents support, first recombinase mixture in the present invention has determined that reconstruction experiment needs the functionally active possessed, the organized enzyme of screening experiment corresponding function again, then determine from function by a large amount of functional verification and express organized enzyme cost all meeting long-range exploitation.In recombinase mixture, the ratio of enzyme is the basic consumption being realized respective function by various enzyme, and on this basis by a large amount of similar proportions parallel laboratory tests, determines optimization system.The present invention compensate for conventional cloning methods complex operation, time-consuming, the more high defect of mortality.Meaning of the present invention is to provide a kind of rapid and convenient efficient cloning process for DNA vitro recombination, in Cytological Basis research and industrial and agricultural production, medicines and health protection etc., establish important basis.
Accompanying drawing explanation
fig. 1it is the flow process signal of the method for the seamless clone of external homologous recombination in the present invention
figure;
fig. 2it is the DNA electrophoresis carrying out bacterium colony PCR checking in the present invention's preferred embodiment
figureresult.
Embodiment
The method of the seamless clone of external homologous recombination in the present invention comprises the following steps:
Step one: preparation linearizing cloning vector
Step 2: prepare Insert Fragment PCR primer
Step 3: recombining reaction
Step 4: recombinant products transforms
Step 5: clone identification
Linearized vector in step one comprises single endonuclease digestion or double digestion by digestion with restriction enzyme, or Inverse PCR amplification obtains.
1. enzyme cutting is for linearizing cloning vector
Use double digestion, linearizing is complete, transform background and false positive clones low.
2. Inverse PCR amplification prepares linearizing cloning vector
Cannot be prepared by PCR when treating cloned sequence to there is a large amount of tumor-necrosis factor glycoproteins, can be increased to pre-linearizing plasmid vector by exo+ polymerase, cloned sequence homologous fragment is treated in two ends interpolation, if PCR primer electrophoretic band is single, amplified production can be directly used in recombining reaction without the need to purifying.
Cloning vector digestion products or pcr amplification product DNA purity lower and likely containing non-linearizing cyclic plasmid, directly use when carrying out recombining reaction, recombination efficiency, clone's positive rate all can decrease.Therefore, when carrying out such as being more than or equal to the clone of 5kb compared with large fragment, the high-quality test kit of recommendation carries out glue to linearizing cloning vector and reclaims purifying, to improve DNA purity and to remove the not linearizing circular vectors of a part (its electrophoresis position is different from linearizing cloning vector).The use-pattern of linearizing cloning vector prepared by different modes can be consulted
table 1.
table 1: the use-pattern of linearizing cloning vector
In step 2, the preparation of Insert Fragment PCR primer comprises design of primers and pcr amplification.
1. design of amplification primers
The total principle of design of primers is: introduce linearizing cloning vector terminal homologous sequence by holding at primer 5 ', make Insert Fragment amplified production 5 ' and 3 ' least significant end respectively with the on all four sequence corresponding with linearizing cloning vector two end, its length is 15bp ~ 20bp.
Forward amplimer design:
5 '-upstream vector terminal homologous sequence+gene specific forward extension increasing sequence-3 '
Reverse amplimer design:
The reverse extension increasing sequence of 3 '-gene specific+downstream vector terminal homologous sequence-5 '
Gene specific positive/negative to extension increasing sequence and conventional Insert Fragment positive/negative to amplimer sequence;
Upstream/downstream carrier end homologous sequence is linearizing cloning vector least significant end sequence (for homologous recombination).
2. Insert Fragment pcr amplification
Insert Fragment recommendation exo+ polymerase increases, to reduce the introducing of amplification sudden change.Without the need to considering that product end is with or without A tail, will be removed in regrouping process, there will not be in final carrier.
After PCR terminates, the product that takes a morsel carries out agarose electrophoresis to check amplification output and specificity.Recombining reaction system compatible conventional PCR reaction system.Therefore, if amplification template is not the cyclic plasmid identical with cloning vector resistance, and PCR primer electrophoretic band is single, and amplified production can be directly used in recombining reaction without the need to purifying.
Pcr amplification product DNA purity is lower, and when directly recombining reaction is carried out in use, recombination efficiency, clone's positive rate all can decrease.Therefore, when carrying out the cloning experimentation compared with large fragment being more than or equal to 5kb, the high-quality test kit of recommendation carries out glue to amplified production and reclaims purifying to improve DNA purity.The use-pattern of Insert Fragment amplified production can be consulted
table 2.
table 2: amplified production recommendation mode
Step 3 comprises preparation reaction system and recombining reaction.
1. in ice-water bath, be formulated as follows reaction system.If accidentally liquid is bonded at tube wall, centrifugally it is made to sink at the bottom of pipe by of short duration.
The ratio of recombinase mixture RecE, RecT and Gamma albumen used in recombining reaction system is 1:1:2 (called after HieffCloneEnzyme); The suitableeest cloning vector usage quantity is 0.03pmol; The suitableeest cloning vector and Insert Fragment mol ratio are 1:2, and namely the suitableeest Insert Fragment usage quantity is 0.06pmol.The DNA quality that these mole numbers are corresponding can be obtained by following formula rough calculation:
The suitableeest cloning vector usage quantity=[0.02 × cloning vector base logarithm] ng (0.03pmol)
The suitableeest Insert Fragment usage quantity=[0.04 × Insert Fragment base logarithm] ng (0.06pmol)
2. recombining reaction
1) after system has been prepared, blow and beat gently up and down with pipettor and mix each component several times, avoid producing bubble, not concuss or vortex mixing.
2) 37 DEG C of reaction 30min are placed in.
3), after question response completes, immediately reaction tubes is placed in ice-water bath and cools 5min.
4) reaction product can directly transform; Also-20 DEG C can be stored in, conversion of thawing when needed.
In step 4, the conversion of recombinant products and coated plate comprise following four steps
1) get 10 μ l and cool reaction solution, join in 100 μ l competent cells, mix under flicking tube wall number, place 30min on ice.
2) 42 DEG C of heat shocks 45 ~ 90 seconds, ice-water bath hatches 2min.
3) add 450 μ lSOC or LB substratum, hatch 10min for 37 DEG C and fully recover.37 DEG C are shaken bacterium 45min.
4) get 100 μ l bacterium liquid to be uniformly coated on containing on suitable antibiotic flat board.Flat board is inverted, in 37 DEG C of incubated overnight.
In step 5 the most convenient of clone identification efficiently method be bacterium colony PCR.
With aseptic rifle head or toothpick single bacterium colony chosen and mix to 20 ~ 50 μ lLB substratum, directly get 1 μ l as pcr template.Recommend at least to carry out bacterium colony PCR with a universal sequencing primer thing, the false-positive generation of PCR can be avoided like this.
The residue bacterium liquid of positive for PCR bacterium colony is seeded to containing overnight incubation in suitable antibiotic LB substratum, extracts plasmid and do follow-up qualification.
According to above-mentioned steps, the plasmid identification extracted after the positive bacterium colony of PCR is cultivated, in proved invention, target dna is under the catalysis of recombinase mixture being added with unique restructuring enhancement factor, in vitro can simple and direct directed cloning efficiently in required carrier.
Below embodiments of the invention are elaborated: the present embodiment is implemented under premised on technical solution of the present invention, give detailed embodiment and process, but protection scope of the present invention is not limited to following embodiment.
Embodiment: effect protein BepC construction of eukaryon expression plasmid for expressing
The nucleotide sequence of BepC albumen is:
ATGTTAGAGCATAATTATTTTTATAAAAACAGCGCAACACTGAAGAATAAACATGGCATAAAAAACCCGCGAAAACTGTATGAACGCTGTGCTCATGAGACAGCCAGAGAGGCTGTAAATTTTCGCCTTGAACCGCCACCAGGGAAATTTGATGCCGCTTATCTAAGGACAATTCACTGGTGCCTTTTCCATAAAACTTTTGAATGGGCCGGTGTTACCCGAGATCAGCCCTTTACATTTGAAGATGGCAGCACTGCATGTATGCCAGCTATGCGACCAAAAGGTTATAAGGTTCCTTTTGCTGTCGGTTCACAAATTCAAAGAGAGCTTAAAAAATTAGAACAAAGACTAACCGCGAAGAATAATTTACAAGGCTTATCGCGCCAAGAATTTGCTGCAAATGCTGCTGAAGTTTTTACAGCTCTCGACCACGCGCATCCTTTCAGAAAAGGCAATGGGCGCACACAACGAATGTTTATGGAAAAACTCGGACAAGCGGCAGGCTATAAGATTGATTTTTCTTTGATCACAAAAGAACGCATGACATATGCCAGCATTGAAGCAATGCAACATAACAATCCAGAACCCATGAAAGATCTTTTTGAGGATATCACTCACCCTCAAAAATCCCTTCTTTTAAAGGAATTTATCTCTCAGATGAGAAGCGCTAGACTTGATGAAATTAACAATCATATTGTTTTGGCAGCAAAAGAAGGTGTGACCTATGATGGCATTTATAAAGGTTCTTCAGCTGAAGGTTTTGTTATAGAAGTAGAAGGTGGCACTTTCATCGTCGGGCACAAAGATGATCTTAAGCCAGAGCAAGTGAAAATATTACAGAATGGTGATTTCATCTCCTTTCAGAAAAACAATGTTCAAAACATGAGAGAAACACTTATCCCAAGCGAGATATTAGCGCCTCTCACCAATGAGATCCTTGCCGAAAGGCTCGTAAACCATTGTGGGGTTGAATCATACCGCCATGAAGTCGAGTGTTTATCAAAAATTGTTTATGGCAACACACAAGCATTAAGCCAAATGATTGAGACAATCAACATAGATCCAAGTTTAGGCGAACAGTTTGTCGATCACATTATCCAAAATCCTAAATCAGTTGGTAAGCTTGCCGGGAAAAAAATCCTGGGTCTAAGAAGCCCAGCGCGCAAACGTGCGGAAGAAACTGTTTCACAGCTTAGTGACACACTTAAAAGTTATGCCGATATAGCACATCAGACCATGGCTGACATCATAGAGCAACACTCAAAAGAACAAAGACGCACAGCGCGCTCTGTCGAAAATCCCGGGAAAGACTTGCAAAACCTTTTTGCCTTGTTCCCAGAACAACAAAGAGAAGCCTTGTCTCATTCCCCTACGCTGCAACAACAACTCCATCGTTTTTCGCGTCAATTGCAAAATCGTTTATCATCAGAGGAGCGTCGAGCCATACAAGAAAACGATTGCACAAGACTTTCTTGTCTGCTTGGTGTATCGGCAAGCAAAGCAAAAGACATTGCTCAAATTGTGAAGCATACCAAAGAGGCACAATGTCAGATGCGTACCCTTAAAGTCTGCCGCTCCGCATCGATGGCTCTGACCAGCTAA
Expression plasmid is pCDNA4.0/TO-Strep, and its multiple clone site is classified as through BamH1 and Xho1 double digestion postorder:
GCGAGCTCGGTACCAAGCTTAAG*********CGCCCAAATTTGCCCGGGAGCT wherein * * * * * * * * * represents plasmid backbone sequence.
PCR primer according to Homology-based cloning design is: (underscore part represents homology overlap)
Upstream: 5 '
aACCATGGCTCGAGCGGATCCaTGTTAGAGCATAATTATT3 '
Downstream: 5 '
gGGTTTAAACGGGCCCTCGAGgCTGGTCAGAGCCATCGATGC3 '
PCR condition: 95 DEG C of denaturations 5 minutes, subsequently 32 circulations: 95 DEG C 30 seconds, 56 DEG C 40 seconds, 72 DEG C 30 seconds, finally extend 5 minutes.PCR primer quantitatively, mixes according to mol ratio 2:1 with linearized vector by product after cutting glue and reclaiming.In 20ul cumulative volume, add 4 μ l damping fluids and 2 μ l recombinases, hatch 30 minutes for 37 degree, subsequently 10 μ l reaction solutions are added in 100 μ l competent cells, carry out chemical conversion.After 16 hours, random picking 10 clones carry out bacterium colony PCR checking, and primer is the same, result
as Fig. 2shown in: 10 clones of picking all successfully insert and treat cloned sequence.
More than describe preferred embodiment of the present invention in detail.Should be appreciated that the ordinary skill of this area just design according to the present invention can make many modifications and variations without the need to creative work.Therefore, all technician in the art, all should by the determined protection domain of claims under this invention's idea on the basis of existing technology by the available technical scheme of logical analysis, reasoning, or a limited experiment.
Claims (10)
1. a recombinase mixture, is characterized in that, described recombinase mixture derives from intestinal bacteria Rec homologous recombination enzyme, and described recombinase mixture comprises RecE, RecT and Gamma albumen.
2. recombinase mixture according to claim 1, is characterized in that, the content ratio of described RecE, described RecT and described Gamma albumen three kinds of recombinases is 1:1:2.
3. the seamless clone's of an external homologous recombination method, is characterized in that, said method comprising the steps of:
Step one: preparation linearizing cloning vector;
Step 2: prepare Insert Fragment PCR primer, by holding the terminal homologous sequence introducing described linearizing cloning vector at primer 5 ', make 5 ' of described Insert Fragment PCR primer end and 3 ' end respectively with the on all four sequence corresponding with described linearizing cloning vector two ends;
Step 3: the described Insert Fragment PCR primer obtained in the described linearizing cloning vector obtained in step one and step 2 is carried out recombining reaction under the effect of recombinase mixture as claimed in claim 1 or 2;
Step 4: the recombinant products obtained in step 3 is carried out cell transformation, obtains transformant;
Step 5: the described transformant obtained in step 4 is cultivated, and carries out clone identification, obtain through described clone identification be positive recombinant DNA molecules.
4. the method for the seamless clone of external homologous recombination according to claim 3, is characterized in that, in described step one, described linearizing cloning vector is obtained by digestion with restriction enzyme or Inverse PCR amplification.
5. the method for the seamless clone of external homologous recombination according to claim 4, is characterized in that, described digestion with restriction enzyme is single endonuclease digestion or double digestion.
6. the method for the seamless clone of external homologous recombination according to claim 4, is characterized in that, when adopting the method for described Inverse PCR amplification, uses exo+ polymerase to increase to pre-linearizing cloning vector.
7. the method for the seamless clone of external homologous recombination according to claim 3, is characterized in that, in described step 2, the design of described primer is as follows:
Forward amplimer design:
5 '-upstream vector terminal homologous sequence+gene specific forward extension increasing sequence-3 ';
Reverse amplimer design:
The reverse extension increasing sequence of 3 '-gene specific+downstream vector terminal homologous sequence-5 '.
8. the method for the seamless clone of external homologous recombination according to claim 7, is characterized in that, the length of described homologous sequence is 15bp ~ 20bp.
9. the method for the seamless clone of external homologous recombination according to claim 3, it is characterized in that, in described step 3, when described Insert Fragment PCR primer is more than or equal to 5kb, described linearizing cloning vector and described Insert Fragment PCR primer mol ratio are 1:10.
10. the method for the seamless clone of external homologous recombination according to claim 3, it is characterized in that, in described step 3, when described Insert Fragment PCR primer is less than or equal to 2kb, described linearizing cloning vector and described Insert Fragment PCR primer mol ratio are 1:2.
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Cited By (4)
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| CN106119222A (en) * | 2016-07-04 | 2016-11-16 | 翌圣生物科技（上海）有限公司 | A kind of protease composition for external homologous recombination, test kit and method |
| CN111321160A (en) * | 2018-12-17 | 2020-06-23 | 中国科学院深圳先进技术研究院 | Rapid cloning method based on reverse amplification full-length plasmid and In-fusion connection |
| CN111850022A (en) * | 2020-07-02 | 2020-10-30 | 南京农业大学 | Method for quickly adding or replacing protein tag of target gene |
| CN114015690A (en) * | 2021-11-08 | 2022-02-08 | 南阳师范学院 | Construction method and application of bombyx mori shRNA expression vector |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106119222A (en) * | 2016-07-04 | 2016-11-16 | 翌圣生物科技（上海）有限公司 | A kind of protease composition for external homologous recombination, test kit and method |
| CN111321160A (en) * | 2018-12-17 | 2020-06-23 | 中国科学院深圳先进技术研究院 | Rapid cloning method based on reverse amplification full-length plasmid and In-fusion connection |
| CN111850022A (en) * | 2020-07-02 | 2020-10-30 | 南京农业大学 | Method for quickly adding or replacing protein tag of target gene |
| CN114015690A (en) * | 2021-11-08 | 2022-02-08 | 南阳师范学院 | Construction method and application of bombyx mori shRNA expression vector |
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| CN105087517B (en) | 2019-05-07 |
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