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.

由于杰弗里斯等人。据报道，具有高变小卫星位点的DNA可用于个体识别，DNA分型系统已被开发出来。这些方法必须分析大量（微克）的宝石 DNA，因此将这些方法应用于由降解或极少量 DNA 组成的法医学样本并不总是可能的。聚合酶链式反应 (PCR) 程序为这个问题提供了解决方案，能够将目标 DNA 区域扩增 100,000 倍。然而，人们发现，扩增极少量 DNA（例如单个精细胞）所需的大量 PCR 循环会导致反应效率更低，常常导致 PCR 产物量不足。众所周知，巢式PCR方法可以提高扩增灵敏度，并且可以应用于各种DNA分析。在本研究中，我们改进了DNA纯化或PCR的程序，以尽量减少污染物质的抑制。此外，我们设计了一种新的PCR系统，在第二次PCR中使用半巢式引物组，以提高HLA-DQAI基因的扩增灵敏度。使用半巢式PCR，可以扩增超过2或3pg的模板DNA并正确输入。研究发现半巢式PCR技术可提高扩增反应的灵敏度和效率，并可成功对HLA-DQA1基因进行分型。这对于从含有极少量 DNA 的样本（例如法医或古代 DNA 样本）进行基因分型很有帮助。