一种改良的启动子序列克隆的染色体步查法

利用染色体步行法，从已知DNA序列克隆侧翼未知序列是非常有效的方法之一，但由于所选用的特定限制性内切酶对目标基因组不能酶解成合适大小的片段，因而受PCR扩增能力的局限,往往扩增不出有效产物. 针对这一点，这里我们介绍一种简单有效的改良方法，它包括以下步骤：首先用不同的限制性内切酶(包括平末端和粘性末端) 酶解目标基因组DNA，接着，选择能将基因组酶切成弥散、分布均匀的限制性内切酶，如DraⅠ和HindⅢ，合成相对应的接头；然后，选择弥散的、分布均匀的限制性内切酶的酶解产物，构建成含相应接头的基因组DNA文库，用作PCR的模板；最后，用接头引物和特异引物，通过巢式PCR扩增目的片段，获得了理想的扩增效果.采用改进后的染色体步查法，有效地从较复杂的棉花核DNA中克隆出6个棉花启动子序列.

An Improved Method of Chromosome Walking for Promoter Sequences Cloning

It is important to isolate and characterize unknown DNA sequences flanking known regions, especially regulatory regions. Among all the polymerase chain reaction (PCR)-based methods, ligation-mediated PCR (LM-PCR) is one of the most suitable methods for genomic walking. Here we describe a simple and efficient improved LM-PCR, which includes the following steps. First, genomic DNA is digested with different restriction endonucleases, including blunt-end and sticky-end ones. Secondly, the restriction endonucleases, which can digest genomic DNA evenly with fine distribution, such as DraⅠand Hind Ⅲ, are selected and corresponding adaptors are synthesized. Then constructed libraries of adaptor-ligated genomic DNA fragments are used as PCR templates. Finally, desired DNA products can be generated from the libraries by Nest-PCR with specific primers and adaptor primers. Using this method,six cotton promoters and one rape promoters have been cloned successfully.