HIV-1基因限制性显示片段的制备（英文）

目的　快速分离HIV 1基因片段制备DNA芯片探针。　方法　以Sau3AⅠ酶切HIV基因后 ,将得到的限制性酶切片段两端接上接头。根据酶切位点与接头的序列设计通用引物。在该通用引物的 3’端分别延伸 1个碱基后 ,通过引物间的两两组合 ,将PCR反应分成 10个亚组。纯化各组PCR产物 ,克隆到T载体上。挑取白色菌斑进行快速鉴定后扩大培养阳性克隆、提质粒。以质粒为模板扩增靶片段并测序。　结果　每个亚型得到了十几个 10 0～ 10 0 0bp的HIV基因片段。　结论　限制性显示技术是一种有效的快速分离制备基因片段的方法。

PREPARATION OF HIV-1 GENE FRAGMENTS BY RESTRICTION DISPLAY

Objective To establish a new method of expeditious isolation of HIV-1 DNA fragments for the preparation of DNA chips. Methods The dissociated HIV genes,which were digested with Sau 3A I to produce multiple gene fragments with size suitable for preparation of DNA microarray,were ligated with universal adapters.PCR primers were designed to match the universal adapters (including the restriction endonuclease site sequence)but with one "nesting"base overhanging at the 3'-end.The PCR reactions were divided into ten subgroups,which were performed either with various single primers or with primer combinations.The PCR products were purified and then cloned into the T-vectors.The positive clones were propagated and the plasmids were extracted.The target HIV gene fragments were isolated and sequenced,which were correlated precisely with the prediction of restriction endonuclease analysis. Results Multiple HIV gene fragments,ranging from 0.1 to 1 kb,were prepared for DNA chips.Conclusion Restriction display-PCR(RD-PCR)is an effective method for expeditious isolation of known or unknown gene fragments.