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双拷贝基因及其侧翼序列的克隆是分子生物学中的一个难点。将优化的反向PCR(Inverse PCR,iPCR)与TAIL-PCR相结合,有效地克隆双拷贝基因及其侧翼序列。先用Southern blotting方法确定一种能获得合适长度片段的限制性内切酶,然后用优化的iPCR方法对该酶切产物进行自连和扩增,将2个拷贝的侧翼序列区分开。根据iPCR结果,进一步用TAIL-PCR扩增更远侧翼的序列。利用这套方法,获得了棉花可育胞质和不育胞质线粒体双拷贝atpA基因的所有EcoR I限制片段(2.2～5.1 kb)和HindⅢ限制片段(8.5～11.7 kb),克隆到2个拷贝各自的侧翼序列。研究结果说明,优化的iPCR与TAIL-PCR相结合是克隆双拷贝基因及其侧翼序列的一种高效方法。
Cloning of the double copy gene and its flanking sequences is a difficult point in molecular biology. The optimized reverse PCR (iPCR) was combined with TAIL-PCR to efficiently clone the double-copy gene and its flanking sequences. Southern blotting was used to determine a restriction endonuclease capable of obtaining fragments of the appropriate length. The digested products were then self-ligated and amplified using the optimized iPCR method to distinguish between two copies of the flanking sequence. Based on the iPCR results, the more distal wing sequences were further amplified by TAIL-PCR. Using this method, all EcoR I restriction fragments (2.2-5.1 kb) and Hind III restriction fragments (8.5-11.7 kb) of the double copies of the atpA gene from fertile and cytoplasmic cotton were obtained and cloned into two copies The respective flanking sequence. The results show that the combination of optimized iPCR with TAIL-PCR is an efficient method for cloning the double copy gene and its flanking sequences.