[Improvement and application of methylation-specific polymerase chain reaction].
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.


CONCLUSIONS
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.
Xiao
Wen-Hua
WH
Department of Oncology, The 304th Hospital, General Military Hospital, Beijing 100 037, P.R. China. w_hxiao@hotmail.com
Liu
Wei-Wen
WW
D006528:Carcinoma, Hepatocellular / Q000235:genetics*; D019175:DNA Methylation; D005260:Female; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D016133:Polymerase Chain Reaction / Q000379:methods*