Method for cloning deoxyribonucleic acid (DNA)

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...

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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 maintain the metastable open-circle DNA complex formed between large fragments and large vectors, which makes it difficult to connect large fragments of DNA; it is usually difficult to complete the multi-fragment ligation reaction at one time using conventional molecular cloning methods, which makes multi-fragment ligation often difficult. Need to go through several steps of subcloning process, and even after several steps of subcloning process, the target gene is often inserted into the vector in the opposite direction, causing false positives

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