Digital Droplet PCR for Monitoring Tissue-Specific Cell Death Using DNA Methylation Patterns of Circulating Cell-Free DNA

Ruth Shemer*, Judith Magenheim, Yuval Dor

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

15 Scopus citations

Abstract

Cell death involves the release of short DNA fragments into blood, termed circulating cell-free DNA (cfDNA). Sequencing of cfDNA in the plasma has recently emerged as a liquid biopsy for detecting fetal chromosomal aberrations, tumor DNA, and graft rejection. However, in cases where cfDNA is derived from tissues with a normal genome, its primary sequence is not informative regarding the tissue of origin. We developed a method of determining the tissue origins of cfDNA, allowing inference of tissue-specific cell death, based on tissue-specific methylation patterns. We have previously described a version of the method that uses next generation sequencing (NGS) to determine methylation patterns in specific marker loci. Here we describe a rapid and simple procedure for cfDNA methylation analysis using droplet digital PCR (ddPCR) on bisulfite treated cfDNA to accurately count the number of molecules carrying a specific methylation signature. Specificity and sensitivity of the assay increases by simultaneously interrogating four to six cytosines in the same molecule using two fluorescent probes. cfDNA methylation analysis using ddPCR can find multiple applications in the non-invasive study of human tissue dynamics in health and disease.

Original languageAmerican English
Article numbere90
JournalCurrent Protocols in Molecular Biology
Volume127
Issue number1
DOIs
StatePublished - Jun 2019

Bibliographical note

Publisher Copyright:
© 2019 John Wiley & Sons, Inc.

Keywords

  • DNA methylation
  • bisulfite
  • blood test
  • cell-free DNA
  • cfDNA
  • ddPCR

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