等位基因特异性PCR方法检测炭疽芽胞杆菌致死因子基因点突变的评价

Evaluation of allele specific polymerase chain reaction applied to point mutagenesis in the lef gene of Bacillus anthracis

目的建立并评价等位基因特异性聚合酶链反应(PCR)在筛选炭疽芽胞杆菌致死基因点突变中的作用。方法等位基因特异性PCR方法。结果等位基因特异性PCR扩增的筛选方法假阳性率较高,其特异性与引物3′端的碱基类型关系不大,与退火温度、模板浓度等均有关系。高保真Taq DNA聚合酶不能改善这种假阳性。结论等位基因特异性PCR筛选方法影响因素较多,假阳性率高,不适于直接作为点突变的筛选方法。

Objective To evaluate the allele specific polymerase chain reaction （AS-PCR） method applied to screen point mutagenesis in lef gene of Bacillus anthracis. Methods AS-PCR amplification. Results The false positive rate of AS-PCR was high, its accuracy was not correlative to the base type at the 3＇ end of primers, and was correlative to annealing temperature and template concentration. The Taq DNA polymerase with proofreading activity couldn＇t improve the false positive. Conclusion AS-PCR amplification could be affected by many factors and is not adapt to be a screen method for point mutagenesis.