Assaying DNA methylation based on high-throughput melting curve approaches

Dayna T. Akey, Joshua M. Akey, Kun Zhang, Li Jin

Research output: Contribution to journalArticle

24 Scopus citations

Abstract

Here we describe two high-throughput methods to assay DNA methylation, melting curve methylation specific PCR (McMSP) and melting curve combined bisulfite restriction analysis (McCOBRA), which adapt standard MSP and COBRA methods to a melting curve analysis based platform. We show that McMSP and McCOBRA can accurately determine methylation status in a high-throughput and gel-free manner. Moreover, McCOBRA can be used to quantitatively estimate the percent of methylated DNA at a specific CpG site within a heterogeneous sample. The accuracy of McMSP and McCOBRA was initially tested using the 5′-CpG site of the tumor-suppressor gene CDKN2A as a model system in homogeneous and heterogeneous controls, and cancer cell line samples. Furthermore, the robustness of McMSP and McCOBRA was validated in four additional loci. We demonstrate that McCOBRA and McMSP provide several advantages over existing methods, as they are simple, accurate, and high-throughput, which makes them widely applicable to large-scale methylation studies.

Original languageEnglish (US)
Pages (from-to)376-384
Number of pages9
JournalGenomics
Volume80
Issue number4
DOIs
StatePublished - Jan 1 2002
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Genetics

Keywords

  • DNA methylation
  • Methodology
  • Sodium bisulfite

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