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DNA polymorphic analysis from extremely small amounts of DNA samples using a semi-nested PCR

使用半巢式 PCR 对极少量 DNA 样本进行 DNA 多态性分析

基本信息

批准号：
04670352
负责人：
KATSUMATA Yoshinao
金额：
$ 1.34万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1993
项目状态：
已结题

项目摘要

Since jeffreys et al. reported that DNA with hypervariable minisatellite loci could be used for identification of individuals, DNA typing systems have been developed. Large amounts(micrograms)of gemonic DNA must be analyzed in these methods, so application of these methods to forensic science samples, consisting of degraded or very small amounts of DNA, is not always possible. The polymerase chain reaction(PCR)procedure has provided a solution to this problem, enabling the amplification of a target DNA region by 100,000 times. However, one finds that the large number of PCR cycles required to amplify extremely small amounts of DNA such as a single sperm cell makes for even less efficiency of the reaction, often resulting in insufficient amounts of PCR products. The nested PCR method is known to increase the amplification sensitivity and can be applied to various DNA analyzes.In the present study, we have improved the procedure of DNA purification or PCR to minimize the inhibition of the contaminated substances. Furthermore, we devised a new PCR system with a semi-nested set of primers in the second PCR to improve the amplification sensitivity of HLA-DQAI gene.Using the semi-nested PCR, more than 2 or 3 pg of template DNA could be amplified and typed correctly. The semi-nested PCR technique was found to enhance the sensitivity and efficiency of the amplification reaction and allowed the successful typing of the HLA-DQA1 gene. This is helpful for genotyping from samples with extremely small amounts of DNA, such as forensic or ancient DNA samples.

自从jeffreys等报道具有高变小卫星基因座的DNA可用于个体鉴定以来，DNA分型系统已经被开发出来。在这些方法中必须分析大量（微克）的基因组DNA，因此将这些方法应用于由降解或非常少量的DNA组成的法医学样品并不总是可能的。聚合酶链式反应（PCR）程序已经提供了解决该问题的方案，使得能够将靶DNA区域扩增100，000倍。然而，人们发现，扩增极少量的DNA（如单个精子细胞）所需的大量PCR循环使得反应效率更低，通常导致PCR产物的量不足。巢式PCR方法可以提高扩增灵敏度，适用于各种DNA分析，本研究对DNA纯化或PCR方法进行了改进，以减少污染物质对扩增的抑制作用。为了提高HLA-DQAI基因扩增的灵敏度，我们设计了一套新的PCR体系，在第二次PCR中采用半套式引物，可扩增出2 ~ 3 pg以上的模板DNA，并能正确分型。半巢式PCR技术提高了扩增反应的灵敏度和效率，并使HLA-DQA 1基因的分型成功。这有助于对含有极少量DNA的样本（例如法医或古代DNA样本）进行基因分型。