Method for cloning deoxyribonucleic acid (DNA)

A DNA cloning and carrier technology, applied in the field of DNA cloning, can solve problems such as false positives, difficulty in connecting large fragments of DNA, and increased screening workload, etc., and achieve the effects of short reaction time, improved cloning success rate, and high positive cloning rate

Inactive Publication Date: 2012-01-18
XIAMEN UNIV
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Problems solved by technology

Furthermore, since the conventional molecular cloning method to connect the vector and the target DNA fragment depends on the ligation reaction catalyzed by ligase, the self-ligation probability of the vector is increased, and the screening workload is increased.
In addition, it is difficult for traditional cloning methods to insert long fragments of DNA into a large molecular weight carrier, mainly because the cohesive ends obtained by restriction endonuclease treatment generally only contain four or five bases. It is difficult to mainta

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  • Method for cloning deoxyribonucleic acid (DNA)
  • Method for cloning deoxyribonucleic acid (DNA)
  • Method for cloning deoxyribonucleic acid (DNA)

Examples

Experimental program
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Effect test

Embodiment 1

[0048] In this example, the human gene PRAK was inserted into the BamHI and XhoI sites of the viral vector pBOB. The specific operations are as follows:

[0049] 1) Digest the circular vector pBOB with BamHI and XhoI to obtain a linear vector with 5'protruding ends at both ends ( figure 1 );

[0050] 2) Design two primers based on the sequence of the target gene hPRAK and pBOB at the upstream 5'end of BamHI and the downstream 3'end of XhoI to amplify the target gene. The two primers are 35 bp in length. The 5'end contains the 15bp vector pBOB homologous sequence (indicated by capital letters), and the 3'end contains the 20bp target gene hPRAK homologous sequence (indicated by lowercase letters).

[0051] hPRAK forward primer sequence: 5'-AGA GAA TTC GGA TCC atgtcggaggagagcgacat-3';

[0052] (The uppercase letters are the homologous sequence at the upstream 5'end of the pBOB vector BamHI, and the lowercase letters are the forward amplification primers at the 5'end of the target gene)

...

Embodiment 2

[0067] The basic steps are the same as in Example 1, the difference is:

[0068] 1) The insertion site of the target gene in the vector pBOB is SmaI. The linear vector generated by the restriction enzyme SmaI in step 1 has blunt ends at both ends;

[0069] 2) The two primers designed by step 3 for PCR amplification of hPRAK are 35 bp in length. The 5'end contains 15 bp vector pBOB homologous sequence (indicated by capital letters), and the 3'end contains 20 bp target gene hPRAK homologous sequence (using Lowercase letters).

[0070] hPRAK forward primer sequence: 5’-TCC AAT ATT CCC GGG atgtcggaggagagcgacat-3’;

[0071] (The uppercase letters are the homologous sequence at the 5'end of the upstream of pBOB vector SmaI, and the lowercase letters are the forward amplification primers at the 5'end of the target gene)

[0072] hPRAK reverse primer sequence: 5'-TGG CTC GAG CCC GGG ttattgggattcgtgggacg-3'.

[0073] (Uppercase letters are the homologous sequence at the downstream 3'end of pBOB ...

Embodiment 3

[0075] The basic steps are the same as in Example 1, except that: the length of the primer designed in step 3 can be changed, and the length of the homologous sequence of the 5'end of the primer can be between 8-20 bp.

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Abstract

The invention relates to a method for cloning deoxyribonucleic acid (DNA). The method comprises the following steps of: digesting a carrier by using a restriction enzyme at an insertion site of the carrier so as to obtain DNA of a linear carrier; synthesizing a polymerase chain reaction (PCR) amplification primer according to the sequence design of a target DNA fragment and the insertion site of the carrier, and performing PCR amplification to obtain the target DNA fragment; purifying the DNA of the linear carrier and the target DNA fragment, mixing, adding exonuclease III and an exonuclease III reaction buffer solution to form a reaction system, continuing to react, adding ethylene diamine tetraacetic acid to terminate the reaction, reacting, and annealing; taking competent cells of transformed E. coli from the reaction system; coating a transformed bacterial liquid on a plate of an Luria-Bertani (LB) culture medium containing corresponding antibiotics and culturing; and selecting a monoclonal bacterial colony from the culture plate, and extracting a plasmid and identifying to confirm the successful cloning of the target DNA fragment. Independent of restriction enzyme cutting sites and ligase, the quick and efficient cloning of DNA fragments can be realized.

Description

Technical field [0001] The invention relates to a DNA cloning method, in particular to an efficient cloning method of target DNA fragments mediated by exonuclease III without ligation reaction. Background technique [0002] The molecular cloning methods currently used in genetic engineering rely on restriction endonucleases and ligases, that is, first obtain target DNA fragments and vector DNA fragments with complementary sticky ends or blunt ends by restriction endonucleases; , The vector and the target DNA fragment are re-linked into a circular plasmid DNA through the ligase ligation reaction, and finally the circular plasmid DNA is transformed into Escherichia coli to obtain a clone containing the target DNA fragment. This conventional molecular cloning method relies on restriction endonuclease recognition sequences, because after being cut by restriction endonucleases, vector DNA and target DNA fragments must produce complementary sticky ends or blunt ends in order to complet...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/66C12R1/19
Inventor 韩家淮梁耀极蒙丹陈万泽吴秀榕李立胜
Owner XIAMEN UNIV
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