Application of multiplex bisulfite PCR-LDR-qPCR assay in interrogating bioinformatically-identified, blood-based methylation markers for colorectal cancer

Colorectal Cancer

Bacolod MD, et al. J Mol Diagn 2020.


The analysis of CpG methylation in circulating tumor DNA (ctDNA) fragments has emerged as a promising approach for the non-invasive early detection of solid tumors, including colorectal cancer (CRC). The most commonly employed assay involves bisulfite conversion of ctDNA, followed by targeted PCR, then qPCR (also known as methylation-specific PCR). This report demonstrates the ability of a multiplex bisulfite PCR-LDR-qPCR assay to detect 7 methylated CpG markers (CRC- or colon-specific), in both

simulated (approximately 30 copies of fragmented CRC cell line DNA mixed with roughly 3000 copies of fragmented peripheral blood DNA), and CRC patient-derived cell-free DNAs (cfDNAs). This scalable assay is designed for multiplexing and incorporates steps for improved sensitivity and specificity, including the enrichment of methylated CpG fragments, ligase detection reaction, the incorporation of ribose bases in primers, and use of uracil DNA glycosylase. Six of the 7 CpG markers (located in promoter regions of PPP1R16B, KCNA3, CLIP4, GDF6, SEPT9, and GSG1L) were identified through integrated analyses of genome-wide methylation datasets for 31 different types of cancer. These markers were to CpG sites at the promoter region of VIM, which along with SEPT9, are markers that have been previously identified to be epigenetic markers of CRC. Additional bioinformatics analyses show that the methylation at these CpG sites negatively correlates with the transcription of their corresponding genes.