甲基化特异多聚酶链反应的改进及应用

Improvement and Application of Methylation-specific Polymerase Chain Reaction

背景与目的:甲基化特异多聚酶链反应(methylation-specificPCR,MSP)已经成为筛查DNA甲基化异常的最常用方法之一,但其方法繁琐、耗时、非特异性产物多和不经济,有待改进。RUNX3基因是胃癌的抑癌基因,失活的主要机制之一为DNA高甲基化,本研究目的在于用改进的MSP检测肝细胞癌RUNX3基因的甲基化状态。方法:采用玻璃奶回收化学修饰后的DNA,应用专用于GC丰富序列扩增的GC-Melt试剂以改良MSP并对152例肝细胞癌RUNX3基因的甲基化状态进行检测,并与常规MSP方法进行比较。结果:常规MSP方法纯化和回收DNA需要昂贵的纯化柱和高速低温离心机以及16h以上的纯化和回收时间;而在改良方法中,玻璃奶回收化学修饰后的DNA不需昂贵的纯化柱和高速低温离心机,纯化和回收过程只需1h。专用于扩增GC丰富序列的GC-Melt溶液较常规方法明显减少PCR的非特异性产物。51.9%(79/152)的肝细胞癌RUNX3基因呈现高甲基化状态。结论:改良的甲基化特异的PCR技术是筛查DNA甲基化高效、简便的方法。甲基化机制可能是肝细胞癌RUNX3基因失活的重要机制之一。

BACKGROUND &OBJECTIVE: Methylation specific polymerase chain reaction (MSP) is frequently used to screen DNA methylation state, but it is complicate and time consuming, with high output of non specific polymerase chain reaction (PCR) products, and diseconomy, these methods need to be improved. RUNX3 gene is a tumor suppressor gene (TSG) in gastric cancer, and inactivated by both hypermethylation and allelic loss. This study was to adopt the improved MSP to detect methylation state of RUNX3 gene in hepatocellular carcinoma (HCC). METHODS: Glassmilk was used to purify and recover DNA modified by sodium of bisulfite; GC Melt solution was particularly used for amplification of GC rich sequence to improve PCR specification. Methylation state of RUNX3 gene in 152 specimens of HCC was detected by this improved MSP, conventional MSP was used as control. RESULTS: In conventional MSP, expensive spin columns, and high speed and low temperature centrifuge should be used to purify and recover DNA, taking up at least 16 h in this process. On contrast, in improved MSP, purifying and recovering DNA with glassmilk need no expensive reagent or equipment mentioned above, taking up only about 1 h in the whole process. Compared with conventional MSP, non specific PCR products of RUNX3 gene were greatly decreased by GC Melt solution. Hypermethylation of RUNX3 gene detected in 51.9%(79/152) of HCC specimens. CONCLUSION: Improved MSP is a rapid, simple, and effective way to screen DNA methylation. DNA methylation may be an important mechanism of inactivation of RUNX3 gene in HCC.