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

随着基因工程和蛋白工程的发展,寡核苷酸指导的基因定位突变技术的应用越来越广泛。但在实际应用的过程中,筛选出所设计的突变克隆仍是比较耗时的。虽然,突变的理论值达50％,而在实际工作中则远远低于此值。造成这种现象的原因很多,包括突变引物的设计是否合理,退火及合成是否完全,模板制备是否符合标准以及M13系统正链转染特性等。过去,人们为提高突变率曾对此方法进行过多次改进。例如,为了富集含突变引物的双链,有用S1外切酶消化法、碱性蔗糖超速离心法、氯化铯密度梯度离心法、溴化乙碇存在下的凝胶电泳,以及用羟基磷灰石吸附单链,等等。但是,这些方法都比较复杂,流程又长。近来,PCR技术已经用于基因定位突变,并可将突变率提高至99％以上,但突变位点和突变方式的随意性仍有一定局限。

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.