一种快速·有效分离T-DNA插入位点的侧翼序列的方法——DW-ACP-PCR技术

A Rapid and Efficient Method of Isolating the Flanking Sequence of T-DNA Inserted Site-DW-ACP-PCR Technique

DW-ACP-PCR(DNAWalking-Annealing control primer-PCR)是一种分离已知DNA序列的侧翼未知序列的新技术。该技术通过3个嵌套的特异引物分别和ACPC复性控制引物组合,进行3步连续的PCR反应,所有反应可以在1 d内完成。ACP的特殊结构能有效地控制非特异扩增。用DW-ACP-PCR技术扩增7个稻瘟菌T-DNA插入突变体的T-DNA插入位点的侧翼序列,均获得了特异目标片段。

DW-ACP-PCR（DNA Walking Annealing control primer PCR） was a new technique of isolating the unknown flanking sequences in known DNA sequence,in which 3 nested specific primers were resp.combined with ACPC renaturation control primers for 3 step continuous PCR reactions.All the reactions needed only one day to be accomplished.The unique structure of ACP could control nonspecific amplification effectively.It was introduced that DW-ACP-PCR technique was used to amplify the flanking sequences of T-DNA inserted site of 7 T-DNA inserted mutants of Magnaporthe grisea.