应用反向PCR方法进行YAC末端的分离分析

Isolating the Ends of Yeast Artificial Chromosome by Inverse Polymerase Chain Reaction

目的建立有效分离酵母人工染色体（ｙｅａｓｔａｒｔｉｆｉｃｉａｌｃｈｒｏｍｏｓｏｍｅ，ＹＡＣ）末端的方法。方法根据反向ＰＣＲ原理，在分析ＹＡＣ载体ＤＮＡ序列的基础上，选用ＹＡＣ载体上存在切点的限制性内切酶酶解ＹＡＣＤＮＡ，通过连接反应形成环状ＤＮＡ分子，再用与ＹＡＣ载体两末端互补的引物进行扩增，扩增产物经进一步纯化后测序，即可得到ＹＡＣ末端的序列。结果用反向ＰＣＲ方法，分别从７７６Ｅ２、９６４Ｃ５、１１Ｄ８和８Ｄ１共４个ＹＡＣ分离得到各自的左右末端。结论该方法简便、有效。

Objective To establish a highly efficient method for isolating yeast artificial chromosome(YAC) ends.Methods Based on the sequence of YAC vector, the frequent cutting enzymes were used to cleave within the vector and the genomic insert to generate relatively small fragments, which were ligated and subsequently amplified using vector primers that are in inverse orientation. The PCR products were purified and sequenced.Results 8 YAC termini were isolated from YAC 776E2, 964C5, 11D8 and 8D1.Conclusion The results suggest that inverse PCR be an efficient method for isolating YAC termini.