High efficiency genome walking method for flanking sequences of cotton mitochondrial double-copy atpA gene based on optimized inverse PCR and TAIL-PCR

Abstract:

Cloning of flanking sequences of double-copy gene is a challenge in molecular biology. We developed a method to solve this problem by combining an optimized inverse PCR (iPCR) with TAIL-PCR. First, Southern blotting analysis was used to determine a proper restriction enzyme that could obtain proper-length restriction fragments that contained the target gene. Then optimized iPCR was performed to amplify the restriction fragments that contained the separated copies of the gene. Based on the obtained sequences, TAIL-PCR was performed to amplify further flanking regions of the gene. With this method, we obtained all of the EcoR I restriction fragments (2.2–5.1 kb) and Hind Ⅲ restriction fragments (8.5–11.7 kb) of mitochondrial atpA gene in cytoplasmic male sterile (CMS) line and maintainer line of Upland cotton. The results showed that this method was an efficient approach to clone flanking sequences of double-copy gene.

Key words:inverse PCR, TAIL-PCR, cytoplasmic male sterility, cotton, atpA, double-copy gene, flanking sequences