一种构建基因组文库的改进方法

构建基因组文库是一项非常基础和重要的工作。但利用传统方法构建基因组文库存在操作步骤繁琐、背景高等问题。为了解决这些问题，对传统构建文库的方法进行了改进。由于Ear I位点经酶切后突出3个可变碱基，经过设计可使酶切后的两个粘端无法匹配，从而防止载体环化，所以用两个Ear I位点作为克隆位点。基因片段是通过用Sau3AI部分酶切基因组总DNA,并在部分酶切片段的3`凹端补一个碱基G获得，因此片段无法串连或环化。本实验构建了基于上述改进的ARS探针载体pHBM803/Trp，并分别用改进方法和传统方法构建水稻基因组文库进行比较。实验结果表明，经过上述两点改进可使文库质量大大提高。

An Optimized Method for Construction of genomic library

Construction of genomic libraries is basic and important. Because of the laboriousness and high background of traditional methods for constructing genomic libraries, we improved them by overcoming these disadvantages. Two Ear I sites were chosen as the cloning sites, which can produce variable 3-base cohesive ends. Therefore the two overhangs could be devised to prevent a match and to avoid self-ligation of vector. Genomic DNA is cleaved partially with Sau3A I and subsequently incubated with dGTP and Klenow fragment of DNA polymerase Iso the self-ligation of fragments and ligation between them are blocked. In this study, the ARS probe vector (pHBM803/Trp) based on the improved method was constructed and then we constructed the Oryza sativa genomic library separately with the traditional method and improved method and compared them. The result of experiment indicated that the improved method could optimize the quality of library.