Vol.48,No.6,2021
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Cover Story：DNA methylation is an important epigenetic phenomena and plays crucial roles in the gene regulation.
Many studies showed that DNA methylation can be used as clinical diagnostic biomarker. However, the ability to
detect the DNA methylation status quickly and accurately is a prerequisite and key point for clinical application.
By using two kinds of primers which can bind to methylated and unmethylated template respectively, methylation
specific PCR (MSP) can distinguish DNA methylation status and prove to be a feasible and convenient diagnostic
technique in clinical practice. Unlike traditional PCR, MSP mainly has four difficulties: how to enhance the
specificity of binding to primer-methylated/unmethylated template, how to reduce the difference of Tm value of
primer sequences, how to remove false positive amplification and how to improve sensitivity. Though most MSP
primer design tools have proposed various solutions for those difficulties, there are still some defects in
consideration of primer influencing factors, multitasking, prediction of specific amplification in MSP primer
design and evaluation. Therefore, in this study, after deep exploration of existing MSP primer design tools, a
novel MSP primer design and graphic evaluation tool named MethyScan was developed based on the integration
of Bowtie, SAMtools, and BEDTools with Python graphic library Matplotlib and third-party functional libraries
Biopython and Primer3-py. Three functional modules were involved in MethyScan including primer design,
genome indexing and primer evaluation. MethyScan not only has the ability to perform MSP primers design and
Nested primers adaptation, but also can evaluate primers specific/non-specific amplification with the analysis of
primer binding information on four converted genomic templates and graphically displaying of the difference
between non-specific amplification and target. Meanwhile, the comparison of MSP primer design for six potential
biomarkers TFPI-2, NDRG4, CDKN2A, CD44, CASP8, and SDHD in esophageal cancer, colorectal cancer, and
other malignant tumors suggested that MethyScan can not only obtain primers with more CpG sites, but also
obtain primers with same or similar locations to those of other softwares, and the difference of Tm values of
primers is even smaller. As the first MSP primer design tool for graphically displaying specific/non-specific
amplification differences, MethyScan can effectively improve the accuracy of methylation primer design and
provides strong support for the development of clinical DNA methylation detection projects, tests and diagnostic
kits. The download address of MethyScan is: https://github.com/bioinfo-ibms-pumc/MethyScan.
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