一种简便高效的寡核苷酸指导的基因定位突变技术

A SIMPLE AND EFFICIENT METHOD FOR OLIGONUCLEOTIDE DIRECTED SITE-SPECIFIC MUTAGENESIS

The synthesized mutant primer including a new created restriction site and a sequencing primer in the Ml3 system were co-annealed with the original template gene. After the strand extention by DNA polymerase the heterogenous double strand DNA(HDSD)was cut with appropriate restriction enzyme at the flanking regions of the target DNA fragment. The HD-SD was isolated and inserted into the polylinker region at the Ml3/pUC system with the same restriction enzymes. The ligation was done directly in the low melting agarose. The white JM 103 transformants were picked out and the matants were screened by restriction analysis and DNA sequencing. The mutation rate could be up to 50%. This method appeared to be simple, time-saving and very efficient.