Quantitative Detection Of Methylation In Plasma DNA Of Colorectal Carcinoma Patients By Real Time PCR

Objective: The common fluorescent quantitative PCR was modified for eliminating non-specific product generated during polymerase chain reaction and elevating the specific of reaction, and the methylated status of MGMT and TPEF were detected in plasma DNA of colorectal carcinoma patients quantitatively by the modified real time PCR. The copy number of the methylated gene was also calculated. The modified protocol was ensured whether it could be used in quantitative detection of aberrant methylation. We also approached the potential clinical value of aberrant methylated MGMT and TPEF in initial detection of colorectal carcinoma, and the significance of allied detection of abrerrant MGMT and TPEF.Methods: According to the analysis of the melting curve, reaction parameter was optimized. The objective DNA fragments were synthesized which were converted according to the principle of bisulphite modification, and the fragments were used to construct methylated MGMT and TPEF plasmid as the standard. The standard curve was made by the modified protocol using the constructed standard. Finally, aberrant methylated MGMT and TPEF were detected quantitatively using the modified real time PCR, and the copy number was calculated according to the standard curve.Results: In 35 plasma DNA of colorectal carcinoma patients, 10 plasma DNA had methylated MGMT and 21 plasma DNA had methylated TPEF. The positive rate were 28.5% and 60%. 23plasma DNA had methylated MGMT or TPEF, the frequency of methylation in at least one of the loci increased to 65.7% for colorectal cancer when combined with MGMT and TPEF. The concentration of methylated MGMT was 23-184 copies/ml, and the content of methyated TPEF was 13-163 copies/ml.Conclusions: The modified protocol is feasible and reliable in quantitative detection of aberrant methylated MGMT and TPEF from the plasma DNA of colorectal carcinoma patients. Aberrant methylation of MGMT and TPEF would be useful epigenetic bio-mark in initial non-invasive detection of colorectal carcinoma, and the combination of MGMT and TPEF could elevate the sensitivity.