LPA法克隆嗜盐菌Halobacterium species xz515古紫质基因

Halobacterium species xz515是从中国西藏分离到的嗜盐菌，所含古紫质(古细菌视紫红质的简称命名为AR4)具有与其他已知菌视紫红质相反的质子释放和吸收的顺序而引起重视．连接反应介导的PCR扩增法(LPA)是一种克隆部分序列已知的基因的新型克隆方法．LPA法利用“酶切-连接-PCR”之组合，首先选定一组限制性内切酶，各自单独地酶切细菌总DNA样品，然后酶切产物分别与相应的寡聚核苷酸片段接头连接．连接液混合起来，作为扩增未知序列DNA的模板．通过这种方法，克隆到了包括完整AR4基因开放阅读框和0．4kb上游调控区域的总长度1．3kb的DNA片段．实验结果表明LPA法可以代替传统的构建基因组DNA文库的方法，可以快速有效地获得目标基因相关的序列．

Rapid Cloning of an Archaerhodopsin Gene from Halobacterium species xz515 by LPA

Ligation-mediated PCR amplification (LPA) is a novel approach developed for rapid cloning of genes starting from partial known nucleotide sequence. In this report, the application of LPA in the cloning of an archaerhodopsin (AR4) gene from Halobacterium species xz515 (H.sp.xz515) is presented. Bacterial genomic DNA samples were first digested by a group of selected restriction enzymes, separately. The resulting digests were then ligated to one of the three types of specially designed adapters corresponding to the restriction enzymes used for digestion. The ligated DNA samples were mixed together and used as the template for semi-nested PCR amplification of the unknown regions by using the common sequence of the adapters as one of the primers. The full-length AR4 gene was obtained by simply joining the sequences derived by LPA. LPA is ideally suited for rapid cloning of promoter regions of eukaryotic genes departing from its cDNA sequence, as well as routine gene cloning works starting from EST sequences.