Abstract
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.
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Acknowledgments
This work was supported by National Cancer Institute (NCI) RO1CA194663, P30CA15704, UO1CA152756, U54CA143862, and P01CA077852 (WMG); Burroughs Wellcome Fund Translational Research Award for Clinician Scientist (WMG); NIH 2T32DK007742-16 (MY); the Lattner Foundation (WMG), R.A.C.E. Charities (WMG).
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Yu, M., Heinzerling, T.J., Grady, W.M. (2018). DNA Methylation Analysis Using Droplet Digital PCR. In: Karlin-Neumann, G., Bizouarn, F. (eds) Digital PCR. Methods in Molecular Biology, vol 1768. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7778-9_21
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DOI: https://doi.org/10.1007/978-1-4939-7778-9_21
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