PCR定点诱变方法构建BCR-ABL cDNA竞争性PCR内参照物

Constructing a Competitor of BCR-ABL cDNA by PCR Site-Directed Mutagenesis

本实验通过合成与原有下游引物 (B)相似的一条新引物 (B′) ,经聚合酶链反应 (polymerasechainreaction ,PCR)扩增出比原BCR ABLcDNA序列减少了 4个碱基的参照物 ,达到定点诱变的目的。经酶切分析证明 ,两型BCR ABLmRNA均可通过此方法得到相应的cDNA参照物。参照物和待测模板的共扩增产物可通过毛细管电泳达到基线分离 ,证明了其可行性。该参照物可用于慢性粒细胞白血病BCR ABL融合基因的竞争性PCR 。

A competitive mimic of the cDNA of the BCR ABL fusion gene was constructed, and its feasibility was testified by capillary electropheresis (CE). The 4 bp shorter mimic was obtained by PCR amplification using a newly synthesized downstream primer analogous to the former one. Mimics of both types of BCR ABL cDNA were achieved and the validity was verified with restriction endonuclease. And the products of the coamplification PCR could be easily separated by capillary electrophorisis. The mimic can be used to quantitative detection of BCR ABL gene through competitive RT PCR in chronic myeloid leukemia.