DNA Methylation Analysis Using Droplet Digital PCR

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 1768; p. 363
• Main Authors: Yu, Ming, Heinzerling, Tai J, Grady, William M
• Format: Journal Article
• Language: English
• Published: United States 2018
• Subjects: Biomarkers, Tumor - genetics; Biomarkers, Tumor - isolation & purification; Cell Adhesion Molecules - genetics; Colon - pathology; Colonic Neoplasms - diagnosis; Colonic Neoplasms - genetics; Colonic Neoplasms - pathology; DNA Methylation - genetics; DNA, Neoplasm - genetics; DNA, Neoplasm - isolation & purification; Humans; MicroRNAs - genetics; Polymerase Chain Reaction - instrumentation; Polymerase Chain Reaction - methods; Promoter Regions, Genetic - genetics; Sensitivity and Specificity; DNA methylation; MethyLight; Droplet digital PCR; Risk biomarkers; MethyLight ddPCR; Colorectal cancer
• ISSN: 1940-6029
• DOI: 10.1007/978-1-4939-7778-9_21

Droplet digital (ddPCR) is a recent advance in PCR technology that enables the precise detection and absolute quantification of nucleic acid target sequences and that has a range of applications for both research and clinical diagnostic studies. Here, we discuss the parameters important in the design and performance of ddPCR for the detection and quantification of methylated DNA. We provide explicit instructions for conducting methylation specific ddPCR (aka MethyLight ddPCR). We also present an example that demonstrates the sensitivity and precision of the method for detecting methylated DNA in the promoter region of mir342/EVL, a potential DNA methylation biomarker for colon cancer risk. Common technical problems and troubleshooting for conducting successful MethyLight ddPCR assays are also discussed.