Nanopore-based assay for detection of methylation in double-stranded DNA fragments

Jiwook Shim, Younghoon Kim, Gwendolyn I. Humphreys, Ann M. Nardulli, Farhad Kosari, George Vasmatzis, William R. Taylor, David A. Ahlquist, Sua Myong, Rashid Bashir

Research output: Contribution to journalArticlepeer-review

51 Scopus citations


DNA methylation is an epigenetic modification of DNA in which methyl groups are added at the 5-carbon position of cytosine. Aberrant DNA methylation, which has been associated with carcinogenesis, can be assessed in various biological fluids and potentially can be used as markers for detection of cancer. Analytically sensitive and specific assays for methylation targeting low-abundance and fragmented DNA are needed for optimal clinical diagnosis and prognosis. We present a nanopore-based direct methylation detection assay that circumvents bisulfite conversion and polymerase chain reaction amplification. Building on our prior work, we used methyl-binding proteins (MBPs), which selectively label the methylated DNA. The nanopore-based assay selectively detects methylated DNA/MBP complexes through a 19 nm nanopore with significantly deeper and prolonged nanopore ionic current blocking, while unmethylated DNA molecules were not detectable due to their smaller diameter. Discrimination of hypermethylated and unmethylated DNA on 90, 60, and 30 bp DNA fragments was demonstrated using sub-10 nm nanopores. Hypermethylated DNA fragments fully bound with MBPs are differentiated from unmethylated DNA at 2.1- to 6.5-fold current blockades and 4.5- to 23.3-fold transport durations. Furthermore, these nanopore assays can detect the CpG dyad in DNA fragments and could someday profile the position of methylated CpG sites on DNA fragments.

Original languageEnglish (US)
Pages (from-to)290-300
Number of pages11
JournalACS Nano
Issue number1
StatePublished - Jan 27 2015


  • cancer detection
  • methyl-binding protein
  • methylated DNA
  • nanopore
  • single-molecule detection

ASJC Scopus subject areas

  • General Materials Science
  • General Engineering
  • General Physics and Astronomy


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