Extraction and processing of circulating DNA from large sample volumes using methylation on beads for the detection of rare epigenetic events

Brian Keeley, Alejandro Stark, Thomas R. Pisanic, Ruby Kwak, Yi Zhang, John Wrangle, Stephen Baylin, James Herman, Nita Ahuja, Malcolm V. Brock, Tza Huei Wang

Research output: Contribution to journalArticle

Abstract

The use of methylated tumor-specific circulating DNA has shown great promise as a potential cancer biomarker. Nonetheless, the relative scarcity of tumor-specific circulating DNA presents a challenge for traditional DNA extraction and processing techniques. Here we demonstrate a single tube extraction and processing technique dubbed "methylation on beads" that allows for DNA extraction and bisulfite conversion for up to 2. ml of plasma or serum. In comparison to traditional techniques including phenol chloroform and alcohol extraction, methylation on beads yields a 1.5- to 5-fold improvement in extraction efficiency. The technique results in far less carryover of PCR inhibitors yielding analytical sensitivity improvements of over 25-fold. The combination of improved recovery and sensitivity make possible the detection of rare epigenetic events and the development of high sensitivity epigenetic diagnostic assays.

Original languageEnglish (US)
Pages (from-to)169-175
Number of pages7
JournalClinica Chimica Acta
Volume425
DOIs
StatePublished - Oct 1 2013

Keywords

  • Blood-processing protocols
  • Cancer epigenetics
  • Circulating DNA
  • DNA methylation
  • Methylation specific PCR
  • Quantification

ASJC Scopus subject areas

  • Biochemistry
  • Clinical Biochemistry
  • Biochemistry, medical

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