Method for cloning seamless gene

Abstract

The invention discloses a method for cloning a seamless gene without an artificial basic group, comprising the steps of: (1) designing and synthesizing a chimera primer according to sequences of a target gene and a target vector; (2) carrying out a single-step binary bridging coupling long-distance PCR (Polymerase Chain Reaction) to obtain a fusion linear fragment; (3) digesting PCR products and removing methylated template plasmid DNA (Deoxyribose Nucleic Acid) by utilizing DpnI (Diphosphopyridine nucleotide I); (4) transforming an escherichia coli strain DH5 alpha; (5) selecting monoclonal colonies for sequencing and identifying; and (6) confirming the integration situation of recombinant plasmids by utilizing enzyme digestion. By means of the method, special restriction enzyme and ligase as well as alkaline phosphatase are needless and any target gene can be merged into any position of any target vector. The method has the advantages of simple principle, simpleness and convenience in operation, accurate and controllable locus, high positive rate and wide application range.

Description

technical field [0001] The invention belongs to the fields of biochemistry and molecular biology, and relates to a seamless gene cloning method without artificial bases. Background technique [0002] Gene cloning is a basic method for gene identification and biological function analysis, and is one of the most important techniques in modern molecular biology. At present, gene cloning methods mainly include two categories, one is ligase-dependent cloning (LDC) and the other is ligase-independent cloning (LIC). LDC is the most widely used. Its main principle is to use restriction endonucleases that can recognize specific DNA sequences to digest the target gene and the target vector, and then use ligase to connect the fragments to form recombinant DNA molecules (LU, Q.2005.Seamless cloning and genefusion.Trends Biotechnol, vol.23, no.4, p.199-207.). The disadvantages of LDC are also very obvious: (1) the steps of enzyme digestion and ligation are complex and cumbersome, time-...

AI Technical Summary

Problems solved by technology

However, this method requires the synthesis of special primers with modifications, so it is difficult to be effectively promoted and used

Recombinase can be used to produce recombinant DNA through in vitro homologous recombination between the target gene and the target vector, but because the price of recombinase is expensive, it is not a preferred solution from an economic point of view (BUCHHOLZ, F. and BISHOP, M.2001 .LoxP-directed cloning: use of Cre recombinase as a universal restriction enzyme. Biotechniques, vol.31, no.4, p.906-908, 910, 912, 914, 916, 918.)

PCR-mediated gene cloning usually uses “megaprimer” for amplification, but its low amplification efficiency often leads to the failure of cloning, which in turn leads to the need to invest more manpower and material resources in condition optimization (VAN DEN ENT, F. and LOWE, J.2006. RF cloning: astriction-free method for inserting target genes into plasmamids. J Biochem Biophys Methods, vol.67, no.1, p.67-74.; BRYKSIN, A.V. and MATSUMURA, I.2010. Overlap extension PCR cloning: a simple and reliable way to create recombinant plasmamids. Biotechniques, vol.48, no.6, p.463-465.)

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