改进的甲基化特异PCR法在抑癌基因p16检测中的应用

目的 介绍一种ＣｐＧ岛甲基化分析方法，即甲基化特异的ＰＣＲ（ｍｅｔｈｙｌａｔｉｏｎ ｓｐｅｃｉｆｉｃ ｐｏｌｙｍｅｒａｓｅ ｃｈａｉｎ ｒｅａｃｔｉｏｎ，ＭＳＰ）法以及对该法的改进。方法 用亚硫酸氢盐修饰被测ＤＮＡ后，再进行甲基化与非甲基化特异的ＰＣＲ扩增，并对ＭＳＰ法进行改进。应用该法分析了颌面部鳞癌中ｐ１６基因５′ＣｐＧ岛甲基化状态。结果 发现一些颌面部鳞癌组织中有ｐ１６基因５′ＣｐＧ岛的甲基

Detection of methylation status of tumor suppressor gene p16 by methylation specific polymerase chain reaction]

OBJECTIVE: To introduce a new method for studies on methylation status of CpG island: methylation specific polymerase chain reaction (MSP) with some modifications. METHODS: Target DNA was modified by sodium bisulfite, converting all unmethylated, but not methylated, cytodines to uracil, and subsequent amplification with primers specific for methylated versus unmethylated DNA. The status of 5'CpG island of tumor suppressor gene p16 in maxilliofacial squamous cell carcinoma (MSCC) was analyzed by modified MSP assay. RESULTS: To get single strand DNA, the authors denatured the target DNA by heating instead of alkaline treatment. DNA purification kit was also replaced by dialysis. A cheaper restriction enzyme Taq I was used in place of BstU I to distinguish methylated fragment from unmethylated fragment. The 5'CpG island of p16 gene was methylated in some MSCC tumors. CONCLUSION: MSP is a simple, sensitive, and specific method for detecting the methylation status of any CpG-rich region and the modifications make it more simple to perform.