Application of Multiplex Bisulfite PCR–Ligase Detection Reaction–Real-Time Quantitative PCR Assay in Interrogating Bioinformatically Identified, Blood-Based Methylation Markers for Colorectal Cancer

Abstract

The analysis of CpG methylation in circulating tumor DNA fragments has emerged as a promising approach for the noninvasive early detection of solid tumors, including colorectal cancer (CRC). The most commonly employed assay involves bisulfite conversion of circulating tumor DNA, followed by targeted PCR, then real-time quantitative PCR (alias methylation-specific PCR). This report demonstrates the ability of a multiplex bisulfite PCReligase detection reactionereal-time quantitative PCR assay to detect seven methylated CpG markers (CRC or colon specific), in both simulated (approximately 30 copies of fragmented CRC cell line DNA mixed with approximately 3000 copies of fragmented peripheral blood DNA) and CRC patientederived cell-free DNAs. 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 seven CpG markers (located in promoter regions of PPP1R16B, KCNA3, CLIP4, GDF6, SEPT9, and GSG1L) were identified through integrated analyses of genome-wide methylation data sets for 31 different types of cancer. These markers were mapped to CpG sites at the promoter region of VIM; VIM and SEPT9 are established epigenetic markers of CRC. Additional bioinformatics analyses show that the methylation at these CpG sites negatively correlates with the transcription of their corresponding genes.

Feasibility of Methylated CLIP4 in Stool for Early Detection of Colorectal Cancer: A Training Study in Chinese Population

“…Limit of detection ( 19 ) of m CLIP4 test was evaluated with a series of mixtures between fully methylated HCT116 genomic DNA and unmethylated Jurkat genomic DNA at different ratios (0, 6.75, 12.5, 25, 50, 60, and 70 pg fully methylated DNA out of 70 pg total DNA per qPCR reaction). The test was performed in 24 replicates for each mixture.…”

“…Hypermethylation of CLIP4 in plasma has been shown for cancer types such as CRC and gastric cancer. Further studies in multiplex blood-based methylation tests validated its potential as another promising biomarker for CRC (16)(17)(18)(19). However, the performance of methylated CLIP4 (mCLIP4) in stool samples for CRC detection has never been reported.…”

Cell-Free DNA-Methylation-Based Methods and Applications in Oncology

“…A multiplex MethyLight assay was applied to the serum cfDNA for the early detection of epithelial ovarian cancer [ 92 ]. A multiplex quantitative PCR, ligase reaction detection reaction quantitative PCR (LDRqPCR), has been set up to improve MSP on cfDNA and was shown to be able to evaluate seven CpG markers relevant in colon cancer [ 93 , 94 ]. Another PCR approach is methylation sensitive high-resolution melting (HRM) methods.…”

Effects of Buffer Concentration on the Sensitivity of Silicon Nanobelt Field-Effect Transistor Sensors

“…For example, the sensor-detection of trace amounts of cancer markers benefits patients in receiving preventative health care and early-stage treatment that can greatly increase cancer survival rates. Conventional methods of sensing biomolecules use enzyme-linked immunosorbent assays and polymerase chain reactions [ 7 , 8 , 9 , 10 ], which sense antigen or antibody levels and DNA fragments, respectively. Both methods require fluorescent molecule labeling on the sensing targets.…”