Optimizing a methylation-specific polymerase chain reaction technology / 中国职业医学

ABSTRACT

OBJECTIVE: To establish a highly specific methylation-specific polymerase chain reaction( MSP) method.METHODS: Bisulphited conversion hypermethylated human genomic DNA and unmethylated human genomic DNA were used as template and p15 gene as the target gene. The Cp G island of p15 gene promoter was predicted by Methprimer software.The methylated and unmethylated primers for polymerase chain reaction( PCR) of methylated and unmethylated DNA templates were designed. The PCR stage of MSP was divided into normal temperature group( methylation primer and unmethylated primer annealing temperature of 58 and 55 ℃,respectively) and elevated temperature group( methylation primer and unmethylated primer annealing temperature were 60 and 57 ℃ respectively). The PCR reaction system was optimized based on the normal temperature group by increasing the concentration of magnesium ions( Mg2 +) and adding dimethyl sulfoxide( DMSO) in the PCR reaction system. RESULTS: Nonspecific amplification products appeared in normal temperature group in the PCR reaction stage of MSP,indicating false positive and false negative reactions. The non-specific amplification was weakened and the specific amplification was equally reduced in the elevated temperature group. The nonspecific amplification disappeared in the optimized PCR system and normal temperature group,and the false positive and false negative reactions were eliminated completely. CONCLUSION: Setting the normal annealing temperature and the concentration of Mg2 +and DMSO is beneficial to ensure the specificity of MSP results.