Traceless cloning and reorganizing method by means of activity of exonuclease
An exonuclease and activity technology, applied in the field of traceless cloning and recombination, can solve the problems of affecting the experimental results, traces of restriction endonuclease sequences, changing the structure and activity of the target protein, etc.
Inactive Publication Date: 2012-07-25
HANGZHOU NORMAL UNIVERSITY
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Problems solved by technology
There are some problems with this method: First, not all genes have suitable restriction sites, especially when there are only a few restriction enzymes for the cloning sites of some vectors; After cloning or recombination of restriction endonucleases, traces of unnecessary restriction endonuclease sequences will be left. In most cases, the recognition sequences of these restriction endonucleases will also cause some additional amino acid sequences on our target protein. Amino acids may change the structure and activity of the target protein, thereby affecting the experimental results
Method used
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Embodiment 1
[0029] Example 1: Utilize exonuclease activity to clone the coding region (open reading frame, ORF) of SGK (serum / glucocorticoid regulated kinase 2) gene 7.1, detect the exonuclease activity of Pfu, T4DNA polymerase and lambda Exonuclease
[0030] In a 10 μl reaction system, 100 ng of DNA fragments were reacted with 1 U of Pfu DNA polymerase, T4 DNA polymerase, and λExonuclease (Table 1) for 5 min, 10 min, and 30 min, respectively, and then 2 μl of 50 mM EDTA was added to terminate the reaction and electrophoresis was performed.
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The invention provides a traceless cloning and reorganizing method by means of activity of exonuclease, which includes: adding DNA (deoxyribonucleic acid) sections with the length of 10-30bp identical with that of sequence at two ends of an insertion site of a cloning carrier to two ends of a target gene respectively, then reorganizing with the carrier DNA under the action of the exonuclease, manly during preparing sections of the target gene, adding the DNA sections with the length of 10-30bp identical with the sequence of the two ends of the insertion site of the cloning carrier respectively to the 5' ends of an amplimer. The traceless cloning and reorganizing method has the advantages that (1) enzyme digestion for the target gene or the DNA sections is omitted, enzyme digestion sites inside the target gene or the DNA sections can be ignored, and the target gene or the DNA sections are free of limits by limit incision enzyme identification sites on the carrier; and (2) unnecessary sequence is avoided introducing when the target genes and the DNA sections are connected to the carrier, namely the traceless cloning.
Description
(1) Technical field [0001] The invention relates to a traceless cloning and recombination method utilizing exonuclease activity. (2) Background technology [0002] Classical gene cloning and recombination experiments are based on type II restriction enzymes and T4 DNA ligase. Use restriction endonuclease to specifically recognize and digest DNA, and then use T4 DNA ligase to connect different DNA fragments together, so as to achieve the purpose of gene cloning and recombination. There are some problems with this method: First, not all genes have suitable restriction sites, especially when there are only a few restriction enzymes for the cloning sites of some vectors; After cloning or recombination of restriction endonucleases, traces of unnecessary restriction endonuclease sequences will be left. In most cases, the recognition sequences of these restriction endonucleases will also cause some additional amino acid sequences on our target protein. Amino acids may change the ...
Claims
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Login to View More IPC IPC(8): C12N15/66C12N15/70C12N1/21C12R1/19
Inventor 戴忠敏孙淑慧黄浩邱猛生
Owner HANGZHOU NORMAL UNIVERSITY