Quantitative Methylation Analysis Using The Multiplex Real-time PCR With Homo-Tag Assisted Non-Dimer System (HANDs-qMSP)

DNA methylation plays a critical role in keeping the fetation,gene imprinting, cell development and cancer in normal.Increasingly,changes in DNA methylation patterns are used as molecular markers of early diagnosis,prognosis as well as individualized treatment for cancer.Therefore,there is a need for reliable and easy techniques to detect and measure DNA methylation in research and routine diagnostics.We have established a modified quantitative analysis of methylated alleles (quantitative methylation specific PCR with Homo-Tag Assisted Non-Dimer system, HANDs-qMSP) based on multiplex real-time PCR(MethyLight).The CIMP(CpG island methylator phenotype,CIMP),including RUNX3,APC,hMLH1,Vim-29 and Vim-50,from 64 colorectal cancer tissue and the corresponding normal tissue were test with HANDs-qMSP.The results show that methylation frequency of the RUNX3,APC,hMLH1,Vim-29 and Vim-50 were 7.81%(5/64),15.63%(10/64),29.69% (19/64),65.63%(42/64),54.69%(35/64) respectively.The specificities of RUNX3,APC,hMLH1,Vim-29 and Vim-50 are 100.00%(64/64),98.44%(63/64),96.88% (62/64),90.62%(58/64) and 83.74%(54/64).Compared to normal multiplex PCR, HANDs-qMSP has two advantages as follow:(1) 98%cutoff tube will be regarded as the calibrator for relativie quantification. Meanwhile it will availably correct the false negative results due to it can check the efficiency of bisulfite treatment.(2) The HAND system can effectively restraint primer dimer and reduce PCR bia between different genes,this improvement not only contributes to precise quantitative accuracy of HANDs-qMSP whose quantification base line can reach 0.01%,but also cut down the labours and expense of experiment.