应用RD-PCR技术制备HIV基因芯片探针

Preparation of HIV Genechip Probes by RD-PCR Technology

利用限制性显示 (RD PCR)技术快速分离HIV 1基因片段制备DNA芯片探针 .以Sau3AⅠ酶切HIV基因 ,得到许多大小适合芯片的限制性酶切片段 .然后在片段两端接上接头 ,根据酶切位点、接头的序列设计通用引物 .在该通用引物的 3′端分别延伸一个碱基后 ,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 .阳性克隆经鉴定、扩大培养后提取质粒 .以质粒为模板扩增靶片段并进行序列分析 .每个亚型得到了十几个 10 0～ 10 0 0bp的HIV基因片段 .研究表明 ,RD

HIV\|1 gene fragments were isolated expeditiously by RD\|PCR for the preparation of DNA chips. The dissociated HIV genes, which were digested with Sau 3AⅠto 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 site sequence but with one “nesting” base overhanging at the 3′\|terminal. The PCR reactions that were performed for various single primers or primer combinations were divided into ten subgroups. The PCR productions were purified and then cloned into the T\|vectors. The positive clones were propagated and the plasmids extracted. The target HIV gene fragments ranging from 0 1 to 1 kb were isolated and sequenced, which were correlated precisely with the RFLP prediction. RD PCR is an effective method for expediting the isolation of known or unknown gene fragments.