CN113667649A - Reaction mixed reagent for lightning cloning and preparation method and application thereof - Google Patents
Reaction mixed reagent for lightning cloning and preparation method and application thereof Download PDFInfo
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- CN113667649A CN113667649A CN202111049566.XA CN202111049566A CN113667649A CN 113667649 A CN113667649 A CN 113667649A CN 202111049566 A CN202111049566 A CN 202111049566A CN 113667649 A CN113667649 A CN 113667649A
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
Abstract
The invention relates to the technical field of molecular cloning, and particularly discloses a reaction mixed reagent for lightning cloning and a preparation method and application thereof, wherein the reaction mixed reagent for lightning cloning comprises a homologous recombinase with an exonucleolytic function, DNA ligase and a reaction buffer solution; the dosage of the homologous recombinase is 0.06uL-0.18uL, the dosage of the DNA ligase is 0.03uL-0.15 uL/total reaction system, and the balance is reaction buffer solution, wherein the total system is 20 uL; the reaction buffer solution comprises the following components in percentage by mass: Tris-HCl 10-16%, MgCl218‑26％、K35-15% of P, 3-9% of KCl, 10-18% of NaCl, 8-12% of dithiothreitol and 15-30% of glycerol. Invention gramThe method overcomes the defects of the prior art, adopts homologous recombinase to clone the exogenous gene to the target site of the vector, and has the advantages of higher accuracy of the reaction mixed reagent, simplicity, convenient use, rapidness, higher cloning efficiency and stability.
Description
Technical Field
The invention relates to the technical field of molecular cloning, and particularly belongs to a reaction mixed reagent for lightning cloning and a preparation method and application thereof.
Background
DNA cloning and subcloning based on today's biotechnology industry and molecular biology, the market today has a continuous need to link several DNA fragments and clone a target fragment to a target site of a target vector, and the conventional method for introducing a foreign gene into a target site of a vector usually comprises the following steps: (1) firstly, carrying out enzyme digestion on a vector by using one or more restriction enzymes, and linearizing the vector; (2) treatment of the linear vector with alkaline phosphatase prevents recircularization of the vector during ligation; (3) carrying out PCR amplification reaction of exogenous DNA by using the primers; (4) treating the amplified exogenous DNA product with the same restriction exonuclease and purifying; (5) linking the vector with exogenous DNA; (6) the ligation product is transformed into a recipient cell, such as E.coli, and recombinants containing the DNA fragment of interest are selected. The traditional cloning method is tedious and time-consuming, has relatively low cloning efficiency, and is limited by restriction enzyme cutting sites on the vector and the foreign gene.
The current various assembling methods are infinite, have advantages and disadvantages and are most suitable for the field, but the current in-vitro assembling method is expensive in whole price or low in efficiency, and is not suitable for large-scale popularization and application. With the development of the field of synthetic biology, higher requirements are put on cloning technology, and the accurate, efficient, simple and economical cloning method is more beneficial to the development of synthetic biology.
Disclosure of Invention
The invention aims to provide a reaction mixed reagent for lightning cloning and a preparation method and application thereof, overcomes the defects of the prior art, adopts homologous recombinase to clone an exogenous gene to a target site of a carrier, and has the advantages of higher accuracy, simplicity, convenient use, rapidness, higher cloning efficiency and stability.
In order to solve the problems, the technical scheme adopted by the invention is as follows:
a reaction mixed reagent for lightning cloning comprises homologous recombinase with an exonuclease function, DNA ligase and reaction buffer solution;
based on the total system of 20uL, the dosage of the homologous recombinase is 0.06uL-0.18 uL/total reaction system, the dosage of the DNA ligase is 0.03uL-0.15 uL/total reaction system, and the balance is reaction buffer solution.
Further, the reaction buffer comprises the following components in percentage by mass: Tris-HCl 10-16%, MgCl218-26％、K35-15% of P, 3-9% of KCl, 10-18% of NaCl, 8-12% of dithiothreitol and 15-30% of glycerol.
Further, each component in the reaction mixed reagent is allowed to be pre-configured to be concentrated at 3 x, 2 x or 5 x concentration, so that the later addition of DNA is reserved, and the concentration condition does not influence the effect of the assembly reaction.
The invention also provides a preparation method of the reaction mixed reagent for lightning cloning, which comprises the following steps:
(1) synthesizing homologous recombinase;
(2) preparing a reaction buffer solution;
(3) and (3) subpackaging the homologous recombinase, the DNA ligase and the reaction buffer.
Further, the specific synthetic method of the homologous recombinase comprises the following steps:
(1) synthesizing a DNA sequence of the recombinase and a base sequence corresponding to the vector enzyme cutting site;
(2) carrying out enzyme digestion on the DNA fragment by using NcoI and XhoI;
(3) connecting the enzyme-cut fragments by using T4 DNA ligase;
(4) transforming into escherichia coli competent cells, and expressing by shaking for 40 minutes at 37 ℃;
(5) after the transformed E.coli is expressed, the homologous recombinase is obtained by purification.
Further, the reaction system for carrying out enzyme digestion on the DNA fragment is as follows:
the reaction was carried out at 22 ℃ for 60 minutes.
The invention finally protects the application of the reaction mixed reagent for lightning cloning in DNA recombination lightning cloning.
Further, the lightning cloning method comprises the steps of adding homologous recombinase with an exonucleolytic function into a system containing a linear vector and a target DNA fragment, digesting the system on ice for 1-3 min to enable the system and the system to generate 5' ends with homologous sequences, inactivating the enzyme at 70-75 ℃, finally annealing to enable the linear vector and the target DNA fragment to recombine to form a circular plasmid with nicks, transforming the circular plasmid into escherichia coli to repair the nicks and copying the circular plasmid along with a host genome
Compared with the prior art, the invention has the following implementation effects:
1. the reaction cuts the DNA fragments by means of homologous recombinase with an exonucleolytic function, so that the assembly efficiency is improved.
2. The reaction does not need the assistance of a complex enzyme system, and has the advantages of simple system, low cost, convenient operation, easy large-scale high-flux operation, short reaction time, low mutation rate, high reaction efficiency and high fidelity. Can rapidly complete the assembly of the assembled DNA molecules.
3. The assembly accuracy is high, and more than 95% of positive clones can be obtained.
Detailed Description
The present invention will be further described with reference to the following examples, but the present invention is not limited to these examples, and any modification is within the scope of the present invention without departing from the spirit of the present invention.
Example 1
The invention discloses a reaction mixed reagent for lightning cloning, which comprises a homologous recombinase with an exonucleolytic function, DNA ligase and a reaction buffer solution;
based on the total amount of 20uL, the dosage of the homologous recombinase is 0.06uL, the dosage of the DNA ligase is 0.03uL, and the balance is reaction buffer solution.
The reaction buffer solution comprises the following components in percentage by mass: Tris-HCl 10%, MgCl2 18％、K3P5%, KCl 9%, NaCl 18%, disulfide12% of threitol and 28% of glycerol.
The components in the reaction mix reagent are allowed to be pre-configured for concentration at 3 x, 2 x or 5 x, which allows for later addition of DNA, and the concentration conditions do not affect the effect of the assembly reaction.
Example 2
The embodiment discloses a preparation method of a reaction mixed reagent for lightning cloning, which comprises the following steps:
(1) synthesizing homologous recombinase;
(2) preparing a reaction buffer solution;
(3) and (3) subpackaging the homologous recombinase, the DNA ligase and the reaction buffer.
The specific synthetic method of the homologous recombinase comprises the following steps:
(1) synthesizing a DNA sequence of the recombinase and a base sequence corresponding to the vector enzyme cutting site;
(2) carrying out enzyme digestion on the DNA fragment by using NcoI and XhoI;
(3) connecting the enzyme-cut fragments by using T4 DNA ligase;
(4) transforming into escherichia coli competent cells, and expressing by shaking for 40 minutes at 37 ℃;
(5) after the transformed E.coli is expressed, the homologous recombinase is obtained by purification.
Further, the reaction system for carrying out enzyme digestion on the DNA fragment is as follows:
the reaction was carried out at 22 ℃ for 60 minutes.
Example 3
The embodiment discloses an application of the reaction mixed reagent for lightning cloning in DNA recombination lightning cloning, and the specific method comprises the following steps: adding homologous recombinase with an exonucleolytic function into a system containing a linear vector and a target DNA fragment, digesting for 1-3 min on ice to enable the two to generate 5' ends with homologous sequences, then inactivating enzymes at 70-75 ℃, finally annealing to enable the linear vector and the target DNA fragment to be recombined to form a circular plasmid with nicks, and transforming the circular plasmid into escherichia coli to repair the nicks and replicate with a host genome.
Comparative example 1
The raw materials and the mixture ratio of the comparative example are basically consistent with those of the example 1, except that: the amount of homologous recombinase used was 0.12uL and the amount of DNA ligase used was 0.09 uL.
Comparative example 2
The raw materials and the mixture ratio of the comparative example are basically consistent with those of the example 1, except that: the amount of homologous recombinase used was 0.18uL and the amount of DNA ligase used was 0.15 uL.
Comparative example 3
The raw materials and the mixture ratio of the comparative example are basically consistent with those of the example 1, except that: t5 exonuclease 0.03uL and commercially available homologous recombinase 0.03uL were used.
Comparative example 4
The raw materials and the mixture ratio of the comparative example are basically consistent with those of the example 1, except that: the reaction buffer solution comprises the following components in percentage by mass: Tris-HCl 13%, MgCl2 21％、K3P10%, KCl 6%, NaCl 14%, dithiothreitol 10%, and glycerol 26%
Comparative example 5
The reaction buffer solution comprises the following components in percentage by mass: Tris-HCl 16%, MgCl2 26％、K315% of P, 3% of KCl, 10% of NaCl, 8% of dithiothreitol and 22% of glycerol
Verification examples
1. Stability detection
Assembling reaction mixed reagents according to the raw material proportions of the example 1 and the comparative examples 1-5 respectively, storing the reagents for 30 days under different temperature conditions respectively, and detecting positive cloning results obtained by cloning 1kb exogenous DNA fragments into a vector respectively; specific results are shown in table 1:
TABLE 1 statistical table for storage of temperature-influencing factor results
Assembling reaction mixed reagents according to the raw material proportions of the example 1 and the comparative examples 1-5 respectively, storing the reagents at 20 ℃ for 10-60 days respectively, and detecting positive cloning results obtained by cloning 1kb exogenous DNA fragments into a vector respectively; specific results are shown in table 2:
TABLE 2 statistical table of retention time influencing factor results
| Serial number | Day 0 | 10 days | 20 days | 40 days | 60 days |
| Example 1 | >98％ | >98％ | >92％ | >90％ | >74％ |
| Comparative example 1 | >98％ | >98％ | >93％ | >86％ | >72％ |
| Comparative example 2 | >98％ | >96％ | >95％ | >90％ | >76％ |
| Comparative example 3 | >96％ | >83％ | >64％ | >58％ | >52％ |
| Comparative example 4 | >98％ | >95％ | >92％ | >90％ | >70％ |
| Comparative example 5 | >98％ | >95％ | >92％ | >86％ | >71％ |
As is clear from the results shown in tables 1 and 2, the reaction mixture reagent of the present invention was very stable, and 95% of positive clones could be obtained even after cloning a 1kb foreign DNA fragment into pUC19 vector and storing the cloned fragment at 0 ℃ for 30 days. After being placed at 20 ℃ for 30 days, the positive cloning rate is not lower than 92%; after being placed at 30 ℃ for 30 days, 90 percent of positive clones can still be obtained; after standing at 20 ℃ for 60 days, 74% of positive clones were obtained. The reaction mixed reagent applied by the invention is obviously superior to the same type of products on the market in stability.
2. Connection efficiency detection
The reaction mixture reagents were assembled according to the raw material ratios of example 1 and comparative examples 1-5, respectively, and different fragments were recombined under reaction conditions of 20-40 minutes of incubation at 22-25 ℃, and the specific results are shown in table 3:
TABLE 3 statistical table of retention time influencing factor results
| Number of segments | 1 | 2 | 3 | 4 | 5 |
| Example 1 | >98％ | >92％ | >86％ | >82％ | >65％ |
| Comparative example 1 | >98％ | >94％ | >89％ | >85％ | >76％ |
| Comparative example 2 | >96％ | >85％ | >79％ | >71％ | >56％ |
| Comparative example 3 | >83％ | >71％ | >65％ | >60％ | >38％ |
| Comparative example 4 | >95％ | >89％ | >81％ | >76％ | >62％ |
| Comparative example 5 | >95％ | >82％ | >73％ | >68％ | >56％ |
As is clear from the results shown in Table 3, the reaction mixture of the present invention was very stable, and when different numbers of foreign DNA fragments (all 1kb) were cloned into the vector, the recombination efficiency of a single fragment was 95% or more, the recombination efficiency of two fragments was 92% or more, the recombination efficiency of 3 fragments was 85% or more, the recombination efficiency of 4 fragments was 80% or more, and the recombination efficiency of 5 fragments was 65% or more. The reaction mixed reagent applied by the invention is obviously superior to the same type of products on the market in recombination efficiency.
The foregoing is merely exemplary and illustrative of the present inventive concept and various modifications, additions and substitutions of similar embodiments may be made to the specific embodiments described by those skilled in the art without departing from the inventive concept or exceeding the scope of the claims as defined in the accompanying claims.
Claims (8)
1. A reactive mixed reagent for lightning cloning, characterized in that: comprises homologous recombinase with an exonuclease function, DNA ligase and reaction buffer;
based on the total system of 20uL, the dosage of the homologous recombinase is 0.06uL-0.18 uL/total reaction system, the dosage of the DNA ligase is 0.03uL-0.15 uL/total reaction system, and the balance is reaction buffer solution.
2. A reactive mixing reagent for lightning cloning according to claim 1, characterised in that: the reaction buffer solution comprises the following components in percentage by mass: Tris-HCl 10-16%, MgCl218-26％、K35-15% of P, 3-9% of KCl, 10-18% of NaCl, 8-12% of dithiothreitol and 15-30% of glycerol.
3. A reactive mixing reagent for lightning cloning according to claim 1, characterised in that: the components in the reaction mixing reagent are allowed to be pre-configured to be concentrated at 3 x, 2 x or 5 x concentration, so that the later addition of DNA is reserved, and the concentration condition does not influence the effect of the assembly reaction.
4. A method of preparing a reactive hybrid reagent for lightning cloning according to claim 1, wherein: the method comprises the following steps:
(1) synthesizing homologous recombinase;
(2) preparing a reaction buffer solution;
(3) and (3) subpackaging the homologous recombinase, the DNA ligase and the reaction buffer.
5. A method of preparing a reactive hybrid reagent for lightning cloning according to claim 4, wherein: the specific synthetic method of the homologous recombinase comprises the following steps:
(1) synthesizing a DNA sequence of the recombinase and a base sequence corresponding to the vector enzyme cutting site;
(2) carrying out enzyme digestion on the DNA fragment by using NcoI and XhoI;
(3) connecting the enzyme-cut fragments by using T4 DNA ligase;
(4) transforming into escherichia coli competent cells, and expressing by shaking for 40 minutes at 37 ℃;
(5) after the transformed E.coli is expressed, the homologous recombinase is obtained by purification.
6. A method of preparing a reactive hybrid reagent for lightning cloning according to claim 5, wherein: the reaction system for carrying out enzyme digestion on the DNA fragment is as follows:
the reaction was carried out at 22 ℃ for 60 minutes.
7. Use of the reactive mixture reagent for lightning cloning of claim 1 in DNA recombination lightning cloning.
8. Use according to claim 7, characterized in that: the lightning cloning method comprises the steps of adding homologous recombinase with an exonucleolytic function into a system containing a linear vector and a target DNA fragment, digesting the system on ice for 1-3 min to enable the two to generate 5' ends with homologous sequences, inactivating the enzyme at 70-75 ℃, finally annealing to enable the linear vector and the target DNA fragment to be recombined to form a circular plasmid with nicks, and transforming the circular plasmid into escherichia coli to repair the nicks and replicate along with a host genome.
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| CA2402321A1 (en) * | 2000-03-07 | 2001-09-13 | University Of Guelph | Dna joining method |
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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 |
| CN107760706A (en) * | 2017-10-11 | 2018-03-06 | 吴江近岸蛋白质科技有限公司 | The application of DNA excision enzymes and the method for seamless clone |
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- 2021-09-08 CN CN202111049566.XA patent/CN113667649A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2402321A1 (en) * | 2000-03-07 | 2001-09-13 | University Of Guelph | Dna joining method |
| CN101899467A (en) * | 2009-05-26 | 2010-12-01 | 上海捷瑞生物工程有限公司 | Method for inserting target DNA fragment into vector |
| CN102443596A (en) * | 2011-12-01 | 2012-05-09 | 中国农业科学院作物科学研究所 | Method for cloning target DNA (deoxyribonucleic acid) by utilizing 3'-5'proof reading activity of exonuclease |
| CN104498451A (en) * | 2015-01-06 | 2015-04-08 | 苏州泓迅生物科技有限公司 | Recombinase with nucleic acid exterior contact and single-chain DNA exchange activity and application of recombinase |
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