应用Agilent 2100 Bioanalyzer分析限制性显示技术制备的HIV片段库

Applying Agilent 2100 Bioanalyzer to Analyze Restriction Displayed HIV DNA Fragments

使用Agilent 2 10 0Bioanalyzer分析限制性显示技术 (restrictiondisplay ,RD)制备的HIV片段库 .利用合适的限制酶从质粒上获得HIVB亚型代表株U2 6 94 2全基因cDNA ,然后将目的片段进行Sau3AⅠ消化 ,在消化得到的片段两端加接头 ,利用通用引物进行PCR扩增 ,扩增结果通过琼脂糖凝胶电泳以及Agilent 2 10 0Bioanalyzer两种方法分析 .结果显示 ,Agilent 2 10 0Bioanalyzer比琼脂糖凝胶电泳能更快速、直接和客观地反映RD技术制备的DNA片段的大小以及含量 ,并能对RD PCR过程中片段自身连接以及优势扩增的现象进行直接的监控作用 .

The bioanalyzer represents a new generation of capillary electrophoresis instruments for the plasma DNA separation based on microfluidics.DNA fragments of HIV-1 prepared by restriction display technique were analyzed with Agilent 2100 Bioanalyzer.The whole cDNA of HIV subtype B U26942 was cleavaged into fragments by restriction endonuclease Sau3A I. The fragments were ligated with adapters and amplified by PCR with universal primer. Agilent 2100 Bioanalyzer and conventional agarose gel electrophoresis were then employed to examine the PCR products respectively. The comparison from two methods showed that Agilent 2100 Bioanalyzer could provide more accurate, and semi-quantitive information of HIV fragments than agarose gel electrophoresis.