Multiplex PCR with minisequencing as an effective high-throughput SNP typing method for formalin-fixed tissue
Multiplex PCR with minisequencing as an effective high-throughput SNP typing method for formalin-fixed tissue
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DOI：
10.1002/elps.200600589
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发表时间：
2007-07-01
期刊：
影响因子：
2.9
通讯作者：
Worobey, Michael
中科院分区：
文献类型：
--
作者：
Gilbert, Marcus T. P.;Sanchez, Juan J.;Worobey, Michael
广泛收集的福尔马林固定石蜡包埋（FFPE）组织可用于遗传分析，以便为疾病或宿主/病原体共同作用的遗传基础提供重要见解。我们在此报告了对44个SNP多重基因分型方法的评估，多重PCR与微序列（MPMS），对6个可变DNA质量的档案FFPE样本进行了92个DNA提取，这些样本的时间在9到25年之间。对质量最高的三种提取物，我们发现测定效率接近100%。然而，最低质量的提取物的效率差异很大。在这项研究中，我们证明，虽然直接测量提取物中的DNA浓度对随后的MPMS成功没有提供有用的信息，但通过使用简单的定量实时PCR （qPCR）检测核DNA的DNA质量的初始筛选，和/或核DNA的最大PCR扩增大小的检测，可以在一定程度上先验地确定该检测的成功。MPMS有望在未来FFPE材料的遗传研究中发挥重要作用。它提供了一种简化的方法来检索大量的遗传信息，使用简单的，单一的反应和微量的档案组织/DNA。根据这一证据，我们建议对FFPE标本进行系统筛选可能在未来为了解过去提供有价值的见解。
Extensive collections of formalin-fixed paraffin-embedded (FFPE) tissues exist that could be exploited for genetic analyses in order to provide important insights into the genetic basis of disease or host/pathogen cointeractions. We report here an evaluation of a 44 SNP multiplex genotyping method, multiplex PCR with minisequencing (MPMS), on 92 DNA extractions performed on six archival FFPE samples of variable DNA quality, which date between 9 and 25 years old. On the three extracts with highest quality, we found the assay efficiency to be near 100%. However, the efficiency of the lowest quality extracts varied significantly. In this study, we demonstrate that although direct measures of DNA concentration in the extracts provide no useful information with regard to subsequent MPMS success, the success of the assay can be determined to some degree a priori, through initial screening of the DNA quality using a simple quantitative real-time PCR (qPCR) assay for nuclear DNA, and/or an assay of the maximum PCR amplifiable size of nuclear DNA. MPMS promises to be of significant use in future genetic studies on FFPE material. It provides a streamlined approach for retrieving a large amount of genetic information using simple, single reactions and minute amounts of archival tissue/DNA. In the light of this evidence, we suggest that the systematic screening of FFPE collections may in the future provide valuable insights into the past.