p16基因甲基化特异检测方法的建立及其初步应用  

Establishment and preliminary application of methylation-specific method for detecting p16 gene

作　　者：刘晓颖[1] 汪惠园[2] 鲁云霞[1] 周青[1] 桂淑玉[3] 夏瑞祥[2] 袁凌云[1] 汪渊[1] 

机构地区：[1]安徽医科大学生物化学教研室和分子生物学实验室,合肥230032 [2]安徽医科大学第一附属医院血液内科,合肥230022 [3]安徽医科大学第一附属医院呼吸内科,合肥230022

出　　处：《安徽医科大学学报》2002年第1期18-21,共4页Acta Universitatis Medicinalis Anhui

基　　金：安徽医科大学自然科学基金 (编号 :5 2 2 0 95;990 7;990 4);安徽省自然科学基金 (编号 :990 44312 );安徽省教育厅自然科学基金 (编号 :JL97 0 77)

摘　　要：目的　建立 p16基因中CpG岛的甲基化分析方法 ,即甲基化特异的PCR(methylation specificpolymerasechainreaction ,MSP)方法及其初步应用。方法　用亚硫酸氢钠修饰变性后的被测DNA ,随之进行甲基化、非甲基化特异的PCR扩增。应用MSP法分析白血病患者的白细胞 p16基因5′CpG岛的甲基化变异情况。结果　用加热法变性DNA ,亚硫酸氢盐修饰DNA ,WizardDNA纯化树脂除盐、纯化修饰后的DNA都获得了成功 ,并建立了MSP分析p16基因甲基化的方法。白血病患者的白细胞 p16基因的 5′CpG岛有一定比例的甲基化变异。结论　MSP是一种较为简便。Objective To establish a method for analysis of the methylation status of CpG islands of p16 gene:methylation-specific polymerase chain reaction(MSP),and its preliminary application. Methods The denatured target DNA was modified by sodium bisulfate,which converting all unmethylated,but not methylated,cytosines to uracils,and then was amplified with primers specific for methylated versus unmethylated DNA.The 5′CpG islands of p16 gene in leukemia DNA were analyzed by MSP assay. Results There were some methylated 5′CpG islands in p16 of leukemia patients.The methods to denature DNA by heating,to modify the denatured DNA by sodium bisulfate,and to purify the modified DNA by Wizard DNA purification resin were all successful. Conclusion MSP is a simple,sensitive,and specific method for determining the methylation status of virtually any CpG-rich region.

关 键 词：P16基因 甲基化 检测方法 

分 类 号：R446.9[医药卫生—诊断学]