Hsu I C, Yang Q, Kahng M W, Xu J F
Department of Pathology, School of Medicine, University of Maryland at Baltimore 21201.
Carcinogenesis. 1994 Aug;15(8):1657-62. doi: 10.1093/carcin/15.8.1657.
We have developed a simple and reliable procedure to screen gene mutations using DNA mismatch repair (MR) specific mut Y enzyme of Escherichia coli and thymidine DNA glycosylase from HeLa cells. The mut Y enzyme cleaves A of G/A mismatches in DNA duplex and thymidine glycosylase cleaves T at G/T mismatches. Previously, we showed the determination of G:C-->T:A mutations in the N-ras gene in two human tumor samples with mut Y G/A MR enzyme. As low as 1-2% mutant DNAs in a sample of mutant and wild-type DNA can be detected with a synthetic DNA to create G/A mispairing for the assay. In this paper, we simplify the assay, include G/T MR thymidine glycosylase from HeLa cells and evaluate the application for screening DNA point mutations of p53 and ras genes. In this method, DNA fragments amplified from normal and mutated genes by polymerase chain reaction (PCR) were mixed and annealed to create DNA mismatches for cleavage by mismatch repair enzymes. The cleaved products and the substrates were separated by gel electrophoresis and detected by autoradiography. In theory, the enzymes that cut G/A or G/T mispairs will detect the mutations of G:C-->A:T, A:T-->G:C, G:C-->T:A and T:A-->G:C. Several human tumor samples were examined for p53 or K-ras mutations with G/A and G/T mismatch repair enzymes. The reliability of mutation detection was evaluated by comparing the results with reported mutations or confirmed by DNA sequencing of the same PCR-amplified DNA fragments. Our data showed that, following mismatch repair enzyme cleavage, all mutated DNA samples yielded cleaved products with sizes as expected. In addition, our assay is able to characterize the nature of mutation by 5' end-labeling of 32P on mutant or wild-type DNA fragments. The low background, reliability and the determination of the sites of mutations as well as the types of DNA base changes indicate the advantages of the method over other techniques in testing DNA mutants.
我们开发了一种简单可靠的程序，用于使用大肠杆菌的DNA错配修复（MR）特异性mut Y酶和来自HeLa细胞的胸苷DNA糖基化酶筛选基因突变。mut Y酶切割DNA双链体中G/A错配的A，胸苷糖基化酶切割G/T错配的T。此前，我们展示了用mut Y G/A MR酶测定两个人类肿瘤样本中N-ras基因的G:C→T:A突变。通过合成DNA产生G/A错配用于检测，在突变型和野生型DNA样本中低至1%-2%的突变DNA都能被检测到。在本文中，我们简化了检测方法，加入了来自HeLa细胞的G/T MR胸苷糖基化酶，并评估其在筛选p53和ras基因DNA点突变中的应用。在该方法中，通过聚合酶链反应（PCR）从正常基因和突变基因扩增的DNA片段混合并退火，以产生DNA错配用于错配修复酶切割。切割产物和底物通过凝胶电泳分离并通过放射自显影检测。理论上，切割G/A或G/T错配的酶将检测到G:C→A:T、A:T→G:C、G:C→T:A和T:A→G:C的突变。用G/A和G/T错配修复酶检测了几个人类肿瘤样本中的p53或K-ras突变。通过将结果与报道的突变进行比较或通过对相同PCR扩增的DNA片段进行DNA测序来评估突变检测的可靠性。我们的数据表明，经过错配修复酶切割后，所有突变DNA样本都产生了预期大小的切割产物。此外，我们的检测能够通过对突变或野生型DNA片段进行32P的5'末端标记来表征突变的性质。低背景、可靠性以及突变位点和DNA碱基变化类型的确定表明该方法在检测DNA突变体方面优于其他技术。
