人体组织中碱基取代突变的大海捞针检测和鉴定

Vincent L Wilson , Qi Wei , Kerry R Wade , Midori Chisa , Deidre Bailey , Christopher M Kanstrup , Xiuqin Yin , Chad M Jackson , Barbara Thompson , William R Lee
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引用次数: 35

摘要

背景和诱导生殖系诱变和其他遗传毒性研究由于缺乏足够灵敏的技术来检测小细胞簇或由数百万细胞组成的组织样本中的单个细胞的突变而受到阻碍。最常见的基因改变类型是基因内的。人类和哺乳动物癌症中的绝大多数致癌突变只涉及单碱基替换。我们已经开发出普遍适用的技术,不仅为位点特异性诱变研究提供必要的敏感性和特异性,而且还可以识别点突变。将聚合酶链反应(PCR)和连接酶链反应(LCR)的指数扩增方法与限制性内切酶(RE)酶切相结合,能够在107个野生型等位基因中以一个突变体的敏感性选择性富集和检测人类致癌位点的单碱基替代突变。这些PCR/RE/LCR程序已成功设计并分别用于Ha-ras和p53基因的密码子12和248,这两个基因都含有天然的MspI限制性内切酶识别序列。这些程序也适用于检测和鉴定不包含天然限制性内切酶识别序列的致癌位点突变。利用PCR技术,将HphI位点整合到人类N-ras基因的密码子12/13区域,然后用于在该致癌位点选择性富集突变体。这些检测N-ras基因密码子12碱基置换突变的PCR/RE/LCR方法在相当于106个野生型细胞的DNA存在下具有检测至少一个突变等位基因的敏感性。在9个正常个体中,只有1个外周血白细胞DNA标本显示可观察到的Ha-ras突变,其频率在10 - 5和10 - 6之间。这些用于检测和鉴定碱基取代突变的PCR/RE/LCR技术普遍适用于人类或动物基因组中的几乎任何位点或碱基位点。这些PCR/RE/LCR技术具有可调的DNA量(基因组数量)和灵敏度(10−2至10−7)的附加优势,可用于解决诱变和致癌中广泛的重要问题。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Needle-in-a-haystack detection and identification of base substitution mutations in human tissues

Background and induced germline mutagenesis and other genotoxicity studies have been hampered by the lack of a sufficiently sensitive technique for detecting mutations in a small cluster of cells or a single cell in a tissue sample composed of millions of cells. The most frequent type of genetic alteration is intragenic. The vast majority of oncogenic mutations in human and mammalian cancer involves only single base substitutions. We have developed universally applicable techniques that not only provide the necessary sensitivity and specificity for site specific mutagenesis studies, but also identify the point mutation. The exponential amplification procedures of polymerase chain reaction (PCR) and ligase chain reaction (LCR) have been combined with restriction endonuclease (RE) digestion to enable the selective enrichment and detection of single base substitution mutations in human oncogenic loci at a sensitivity of one mutant in more than 107 wild type alleles. These PCR/RE/LCR procedures have been successfully designed and used for codons 12 and 248 of the Ha-ras and p53 genes, respectively, both of which contain a natural MspI restriction endonuclease recognition sequence. These procedures have also been adapted for the detection and identification of mutations in oncogenic loci that do not contain a natural restriction endonuclease recognition sequence. Using PCR techniques, a HphI site was incorporated into the codons 12/13 region of the human N-ras gene, which was then used for the selective enrichment of mutants at this oncogenic locus. These PCR/RE/LCR procedures for base substitution mutations in codon 12 of the N-ras gene were found to have the sensitivity of detection of at least one mutant allele in the presence of the DNA equivalent of 106 wild type cells. Only one peripheral blood leukocyte DNA specimen out of nine normal individuals displayed an observable Ha-ras mutation that was present at frequency between 10−5 and 10−6. These PCR/RE/LCR techniques for detecting and identifying base substitution mutations are universally applicable to almost any locus or base site within the human or animal genome. With the added advantage of the adjustability of both the amount of DNA (number of genomes) to be tested and the sensitivity (10−2 to 10−7) of the assay selection or enrichment procedures, these PCR/RE/LCR techniques will be useful in addressing a broad range of important questions in mutagenesis and carcinogenesis.

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