石蜡包埋组织DNA提取方法的比较及巢式PCR技术的应用  

Analysis of Different Methods of Extracting DNA from Paraffin-embedded Tissues and the Application of Nest PCR

作　　者：鄢丽敏[1] 孙保存[1] 赵秀兰[1] 刘增辉[1] 宋文静[1] 

机构地区：[1]天津医科大学病理教研室,天津300070

出　　处：《生物医学工程学杂志》2011年第4期795-799,共5页Journal of Biomedical Engineering

基　　金：天津市科技发展基金资助项目(020127)

摘　　要：确定从福尔马林固定、石蜡包埋(FFPE)组织中提取DNA的最佳方法,增强长片段PCR产物的成功扩增率,便于分子生物学与临床医学的有机结合。本文选取正常甲状腺FFPE标本20份,分别应用"一步法"、酚/氯仿抽提法和试剂盒法三种不同的方法提取总DNA,并应用传统PCR法和巢式PCR法对线粒体DNA(mtDNA)的D环区进行扩增。结果酚/氯仿抽提法所得样本DNA的OD260/D280比值最高,为1.703±0.086。"一步法"、酚/氯仿抽提法和试剂盒法提取DNA的普通PCR长片段扩增成功率分别为0%、5%和10%,而巢式PCR长片段扩增成功率分别为0%、95%和85%。可见用蛋白酶K消化、酚/氯仿纯化法提取的FFPE组织标本DNA质量可靠,巢式PCR技术是从石蜡标本中扩增长片段DNA的有效方法。The aim of this research was to explore the most optimal method of DNA extraction from formalin-fixed,paraffin-embedded（FFPE） tissues,and to improve the amplification of long fragments with the method.Three methods,one step method,phenol-chloroform extraction method,and genomic DNA purification kit method,were employed to extract DNA from twenty normal thyroid tissues which were fixed with formalin and embedded with paraffin.The highest proportionality of OD260/OD280 in the examples was obtained by phenol-chloroform extraction method,1.703±0.086,compared to the results of the other two methods.As for the long DNA segments amplification,the achievement ratio of one step method,phenol-chloroform extraction method and genomic DNA purification kit method were 0%,5% and 10%,respectively,by traditional PCR method,but 0%,95% and 85% respectively by the nest PCR.We have found that the best process of extracting DNA from FFPE is digesting by proteinase K and purifying by phenol-chloroform,and it is effective to amplify long DNA segments from FFPE by nest PCR.

关 键 词：石蜡包埋组织 DNA提取 巢式PCR 比较 

分 类 号：R450[医药卫生—治疗学]