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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。
OBJECTIVE: To rapidly isolate HIV 1 gene fragment and prepare DNA chip probe. Methods After the HIV gene was digested with Sau3A Ⅰ, the ends of the obtained restriction fragments were ligated to the linker. Universal primers were designed based on the sequence of the cleavage site and linker. After extending one base each at the 3 ’end of the universal primer, the PCR reaction was divided into 10 subgroups by any two-primer combination. Each PCR product was purified and cloned into T vector. Pick white plaque for rapid identification after expansion of positive clones raised plasmid. The plasmid was used as a template to amplify the target fragment and sequenced. As a result, more than a dozen HIV gene fragments of 10 0 to 100 bp were obtained for each subtype. Conclusion Restriction display technology is an effective method for rapid isolation of gene fragments.