Optimization of PCR conditions in DNA methylation assay

【Objective】To establish a optimal procedure for DNA methlation PCR analysis,the optimized reaction conditions and methods of PCR in the assay has been studied.【Methods】The promoter region in AFP gene of MHCC-97H HCC cell lines were used as the template.The affecting factors in PCR include Mg2+,TaqDNA polymerase,annealing temperature and procedure et al.【Results】In the 20 μl amplification reactions system containing template DNA 2 μl,10×PCR buffer(Mg2+ free) 2.5 μl,MgCl2 2 μl,dNTPs 2.5 μl,Taq DNA polymerase 0.4 μl,sense primer 1 μl and antisense primer 1 μl.The optimal amplified procedure was studied and the Touch Down PCR had been adopted.【Conclusions】A reaction system and amplified procedure suitable for methylation assay were established.It is the foundation for the study of mechanism of tumorigenesis.