用普通琼脂糖代替低熔点胶回收DNA片段

Replacing Low Melting-Temperature Agarose by General Agaroseto Recollect DNA Fragment

为了建立一种直接从普通琼脂糖凝胶中回收DNA片段的简便实用的方法 ,采用聚合酶链式反应扩增人P53基因外显子7、8和其间的内含子7序列 ,用普通琼脂糖凝胶电泳,直接从凝胶中切下产物带 ,用加热熔化法回收DNA ;紫外比色法测定回收率 ;用测序法鉴定回收产物质量。并用QIAquickSpin纯化柱对照。结果表明 ,本法回收的产物质量明显优于用QIAquickSpin柱回收 ,本法回收的产物用于测序效果极佳 ,回收率达80% ,用QIAquickSpin柱回收率不到20% ,差异非常显著(P<0 01)。证明这种方法回收PCR产物质量可靠 ,能代替低熔点胶回收DNA 。

In order to find a simple and efficient method to isolate single or double strand DNA fragment amplified by polymerase chain reaction (PCR),we used PCR method to amplify exon 7,exon 8 and intron 7 of human P53 gene, electrophoresis to identify products,fusion and phenol chlorofom extraction (FPC) to isolate specific DNA from agarose gel,ultraviolet colorimetry to deteminate collected rate,and direct sequencing to identify the quality of recollected DNA. A control test was also made by using QIAquick Spin Colum The results showed that the quality of PCR products recollected by using FPC method was very good When the recollected DNA was used in sequencing,no matter what was single or double strand DNA,the sequence data was clear and even,with low noise The recollected rate of using FPC,which was over 80 per cent, was higher than that of using colum (lessthan 20 per cent), there were statistical significances (P<0 01) In the control test, it had a little non specific DNA in the collected products,and the sequencing experiment of using double strand products was failure All above mentioned suggested that general agarose gelis efficient in place of low melting temperature for isolating DNA fragment