一种实用可靠的古代DNA获取方法  

A Practical and Efficient Method for The Retrieval of Ancient DNA Sequence

作　　者：刘冉冉[1] 袁靖[2] 赵兴波[1,3] 李宁[1] 

机构地区：[1]中国农业大学农业生物技术国家重点实验室,北京100193 [2]中国社会科学院考古研究所,北京100710 [3]中国农业大学动物科技学院,北京100193

出　　处：《生物化学与生物物理进展》2009年第11期1495-1502,共8页Progress In Biochemistry and Biophysics

基　　金：国家重点基础研究发展计划项目(973)(2006CB102100);国家科技支撑计划课题(2006BAK21B03)资助~~

摘　　要：古代DNA序列信息能够为物种演化研究提供最直接的分子证据,但获取古代DNA的技术仍存在诸多瓶颈,尤其是扩增中存在受损伤DNA模板的干扰、获取成本高和实验周期长等问题.改进了异丙醇沉淀提取法,并采用了尿嘧啶糖苷酶(UNG)去除受损伤DNA模板后进行扩增的方法,最终可以高效地获取真实的古代DNA序列.实验利用距今4300～3900年前的猪牙样本,将改进的古DNA获取方法与常规方法进行比较研究,结果表明,改进的异丙醇沉淀法提取结合UNG处理后进行PCR扩增的方法,可以在保证古代DNA获取成功率并提高获得的DNA序列可靠性的前提下,将经费投入和实验周期都各减少至常规方法的50%以下.这可以为开展大规模古代样本检测提供一种切实可行的DNA获取方法.Retrieval of ancient DNA （aDNA） sequences from organism remains provide direct view of their evolutionary history.However,researches on aDNA have suffered from lots of technical problems.Specifically,discredited sequences were generated from damaged aDNA templates,and expensive and time-consuming methods were employed.Here,a method which could recover the endogenous aDNA as well as to reduce the cost and research period is described.This is achieved by improving the ancient DNA extraction method of isopropanol precipitation,and reevaluating the method of PCR after N-glycosylase （UNG） treatment,which could remove the damaged DNA from the aDNA extract.The efficiency of these methods were tested by comparing with traditional methods using ancient specimens of pig teeth aged between 4 300 years before present （BP） and 3 900 BP.The results showed that：firstly,the extraction efficiency of the improved method of isopropanol precipitation and current method with silica-based spin column were all 60%.Furthermore,the research period at least could be reduced by half with the application of the improved methods and the cost to 1/10 of the current method.Secondly,sequences obtained through the method of PCR after UNG treatment were 100% authentic.In contrast,66%～88% sequences were authentic based on the results obtained with the method of multiple PCRs without UNG treatment.And the research cost and period needed by the method with UNG treatment were only half of the later one.These results demonstrate that the improved extraction method of isopropanol precipitation combined with the method of PCR after UNG treatment could increase the success rate of authentic DNA amplified and at least reduce the research cost and period by half.Therefore,this method can be applied in the large-scale detection of ancient specimens.

关 键 词：古代DNA DNA提取 受损伤DNA PCR 

分 类 号：Q751[生物学—分子生物学]