|
用普通琼脂糖代替低熔点胶回收DNA片段 |
| |
顾其华 李玲芝 舒畅 杨志毅 叶爱慧
摘 要：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QIAquick Spin纯化柱对照。结果表明，本法回收的产物质量明显优于用QIAquick Spin柱回收，本法回收的产物用于测序效果极佳，回收率达80％，用QIAquick Spin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，有较大的实用价值。
关键词： 聚合酶链式反应； 热熔化； 苯酚－氯仿； DNA分离
中图分类号： Q781 文献标识码： A 文章编号： 0253－9772(2000)02－0103－03
Replacing Low MeltingTemperature Agarose by General Agaroseto Recollect DNA Fragment
GU Qi-hua1 LI Ling-zhi SHU Chang YANG Zhi-yi YE Ai-hui1
（Laboratory of Molecular Genetics of The First People s Hospitalin Huaihua,Huaihua,418000）
（Xiangya Affiliated Hospital of Hunan Medical University,Changsha 410078，Chian）
Abstract： In order to find a simple and efficient method to isolate single or doublestrand 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 phenolchlorofom 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 doublestrand 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 nonspecific DNA in the collected products,and the sequencing experiment of using doublestrand products was failure．All above mentioned suggested that general agarose gelis efficient in place of low meltingtemp[1] [2] 下一页
上一个医学论文： Clarke
下一个医学论文： 赣中南花猪随机扩增多态DNA与群体遗传关系的研究
|
|
|
|
|
|
|
您现在的位置： 绿色健康网 >> 医学论文 >> 基础医学论文 >> 正文