| Summary: | 碩士 === 高苑科技大學 === 化工與生化工程研究所 === 100 === Screening cancer-specific methylation markers is one of promising tools that could facilitate early cancer detection. In our previous study, we demonstrated that methyl-CpG-binding (MB) PCR is a less expensive method that could reliablly detect the methylated gene in the clinical specimens with limited amounts of DNA. However, before making this method as a cost-effective test for cancer screen, we have to develop an MB-based multiplex PCR (MBmxPCR) that could determine the methylation status of a panel of marker genes in a single test. The optimized MBmxPCR can also be used to evaluate the role of numerous cancer-associated marker gene(s) through examination of the the archieved samples that had been well characterized pathologically.
MBmxPCR followed the protocol we had published elsewhere. MsssI-treated DNA and Phi-29 amplified DNA were used as global methylation and unmethylation control, respectively. Selection of the methylation markers focused on those that were often commonly methylated in the prostate cancers but rarely in the benign prostate tissues. PCR internal controls for either methylation or unmethylation were also included in each MBmxPCR. To simplify the optimization steps of the MBmxPCR, each primers were designed by adding a 20-nucleotide M13 sequence to the 5' end of the gene-specific squences. Testing DNA samples were prepared from 56 archieved paraffin-embedded prostatic tissues, including 28 each with cancerous and benign disease. Bisulfite sequencing analyses were used as gold standards to evaluate the reliability of the optimized MBmxPCR.
Form the series of experiments, the results provided the following conclusions. First, the ideal fragment sizes of the methylated sequences best for the MBmxPCR seem to be in the range between 150 bp and 880 bp. The fragments longer than 880 bp would cause false negative results, while those shorter than 150 bp often led to false positive results. Second, the PCR coverage region of each gene-specific primer should not be shorter than 150 bp, particularly in the examination of the paraffin-embedded tissues. The degraded DNA fragments in such samples would inevitably lead to false positive results. Third, for MBmxPCR, the ideal stringent washes applied before PCR amplification was 300mM NaCl. When using washes with 400mM or 200mM NaCl, the MBmxPCR would greatly decrease in detection sensitivity or specificity, respectively. Four, when examining the degraded DNA samples containing large amount of short fragments, the most reliable MBmxPCR results would rely on fractioally deleting most of the short fragments, followed by appropriate enzymatic cleavage. Five, even under the optimized conditions, the input amount of DNA required for MBmxPCR analysis of the paraffin-embedd tissue samples was 100ng, instead of 5ng or less required for examing the samples with intact DNA fragments. Finally, preliminary of bisulfite sequencing results showed a full concordance to the results disclosed by the optimized MB-mxPCR.
Key words：early cancer detection, methylation, methyl-CpG-binding domain (MBD), multiplex polymerase chain reaction (mxPCR)
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