An endonuclease/ligase based mutation scanning method especially suited for analysis of neoplastic tissue
An endonuclease/ligase based mutation scanning method especially suited for analysis of neoplastic tissue
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DOI：
10.1038/sj.onc.1205109
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发表时间：
2002-03-14
期刊：
影响因子：
8
通讯作者：
Barany, F
中科院分区：
文献类型：
--
作者：
Huang, JM;Kirk, B;Barany, F
已知和候选癌症基因中的遗传性和散发性突变的知识可能会影响临床决策。我们开发了一种结合热稳定内切酶V（Endo V）和DNA连接酶的突变扫描方法。使用荧光标记的引物产生变体和野生型PCR扩增子，并进行杂合。海栖热袍菌（Thermotoga maritima，Tma）EndoV识别并主要切割异源双链DNA的3'端一个碱基的错配，以及以低水平切割匹配的DNA。栖热菌（Thermus species）AK 16 D DNA连接酶重新密封背景切口，以创建高度灵敏和特异性的测定。DNA测序凝胶上的片段迁移率揭示了突变的大致位置。该方法在p53、VHL、K-ras、APC、BRCA 1和BRCA 2基因中分别鉴定了31/35和8/8个独特的点突变和插入/缺失。该方法对检测用野生型DNA以1：20稀释的K-ras突变、1.7 kb扩增子中的p53突变以及合并DNA样本中未知的p53突变具有灵敏度。EndoV/Ligase突变扫描结合PCR/LDR/Universal阵列被证明在检测结肠肿瘤中的p53突变方面优于自动DNA测序的上级。该技术非常适合于扫描合并样本中的低频突变，并分析含有少数未知突变的肿瘤DNA。
Knowledge of inherited and sporadic mutations in known and candidate cancer genes may influence clinical decisions. We have developed a mutation scanning method that combines thermostable EndonucleaseV (Endo V) and DNA ligase. Variant and wild-type PCR amplicons are generated using fluorescently labeled primers, and heteroduplexed. Thermotoga maritima (Tma) EndoV recognizes and primarily cleaves heteroduplex DNA one base 3' to the mismatch, as well as nicking matched DNA at low levels. Thermus species (Tsp.) AK16D DNA ligase reseals the background nicks to create a highly sensitive and specific assay. The fragment mobility on a DNA sequencing gel reveals the approximate position of the mutation. This method identified 31/35 and 8/8 unique point mutations and insertions/deletions, respectively, in the p53, VHL, K-ras, APC, BRCA1, and BRCA2 genes. The method has the sensitivity to detect K-ras mutations diluted 1 :20 with wildtype DNA, a p53 mutation in a 1.7 kb amplicon, and unknown p53 mutations in pooled DNA samples. EndoV/Ligase mutation scanning combined with PCR/LDR/Universal array proved superior to automated DNA sequencing for detecting p53 mutations in colon tumors. This technique is well suited for scanning low-frequency mutations in pooled samples and for analysing tumor DNA containing a minority of the unknown mutation.