METHOD FOR SITE-DIRECTED MUTAGENESIS USING INVERSE PCR

摘要

A kind of method is arranged to change, removes or add a specific matrix of a gene, auxiliary agent or other DNA parts, be cloned in a kind of mixture of 3 kinds of enzymes of a vector sum by using inverse PCR for inducing site-specific mutagenesis by using inverse PCR. A kind of method, include the following steps: for inducing site-specific mutagenesis by using inverse PCR: (i) inverse PCR is executed, by using a prime pair of the opposite direction with the mutagenesis in the desired site with circular double rope DNA molecule as a mould; (ii), the reaction of 3 kinds of enzymes, by increasing a kind of mixture of 3 kinds of enzymes to PCR products. 3 kinds of enzymes include kinases, are used to make 5 ' end phosphorylations of a priming paint; Ligase is used to connect both ends and the restriction enzyme of the PCR products of amplification, for cutting the template strand to have methylated. Mutagenesis is mutagenesis (substitution), deletes or be inserted into.