中国生物工程杂志

Site-directed mutagenesis is a common method in molecular biology research. For single-site mutagenesis,the classic method (overlap extension PCR) and the recently developed methods (including rolling circle replication and megaprimer PCR) have both advantages and disadvantages. For multi-site mutagenesis,single-site mutagenesis with multiple rounds PCR is probably still the best strategy although it is tedious and inefficient. In order to improve the efficiency of multi-site mutagenesis,the classic overlap extension PCR can be improved appropriately. First of all,it is necessary to analyze the relative positions between the two mutation sites and develop different mutation primer design strategies accordingly. If two sites are adjacent to each other,or with a distance less than 15bp,one mutational primer pair which contains both mutation sites can be designed and complete the two sites together. If the distance between two sites is between 15-30bp,two sets of mutational primer pair with each set covering each site are necessary,with overlapping regions between these two sets of primer pair to enable second round PCR covering the two mutation sites. If the distance between two mutant sites is further increased,it is implausible to design one mutational primer to cover both mutation sites and use two sets of primer pair with overlapping regions due to the limited length of DNA synthesis in one chemical reaction. Therefore,a new strategy of multi-site mutagenesis by multi-fragment overlap extension PCR was proposed. This multi-fragment strategy divides the gene into three fragments which can be joined together in the second round of PCR in a multi-fragment overlap extension manner with two pairs of mutant primers with overlapping regions. The multi-fragment overlap extension PCR can be adopted when the distance between the two mutation sites is longer than 90bp,considering the limitation of DNA detection by electrophoresis and the efficiency of purification of DNA fragment from agarose gel. Combining the above strategies about mutational primer designing with multi-fragment overlap extension PCR,it is possible to produce multi-site mutations simultaneously in one mutagenesis cycle. Taking the Rb1 gene as an example,its C-terminal fragment contains several potential phosphorylation sites of CDK2/4. A phosphorylation mutant of Rb1 which is designated as PSM6 contains 6 site-directed mutations on phosphorylation sites of C-terminal of Rb1 gene. To achieve PSM6 mutant of RB1 C-terminal fragment,two pairs of mutant primers were designed using the multi-fragment strategy. In the first round PCR,three fragments were amplified using wild type Rb1 gene C-terminal as template,and the PSM6 mutant were amplified using the three fragments overlap extension product from the first round PCR as templates in the second round of PCR. The DNA product,which was supposed to containing 6 phosphorylated site mutants,was digested by restriction endonuclease and then ligated into corresponding plasmid vector by conventional molecular cloning method. According to the DNA sequencing result,it was found that the inserted fragment of recombinant plasmid contains all 6 phosphorylated site mutants as expected. Therefore,a flexible primer design strategy,combined with multi-fragment overlap extension PCR,can be used to produce multi-site gene mutants with one round of mutagenesis,which is a powerful method for multi-site mutagenesis in the research work.