MS-qFRET: A quantum dot-based method for analysis of DNA methylation

http://genome.cshlp.org/content/early/2009/05/14/gr.088831.108
Vasudev J Bailey1, Hariharan Easwaran1, Yi Zhang1, Elizabeth Griffiths1,
Steven A Belinsky2, James G. Herman1, Stephen Baylin1, Hetty Carraway1 and
Jeff Wang1,3
E-mail: thwang@jhu.edu

Abstract

DNA methylation contributes to carcinogenesis by silencing key tumor suppressor genes. Here we report an ultrasensitive and reliable nanotechnology assay, MS-qFRET, for detection and quantification of DNA methylation. Bisulfite modified DNA is subjected to PCR amplification with primers that would differentiate between methylated and unmethylated DNA. Quantum dots are then used to capture PCR amplicons and determine the methylation status via fluorescence resonance energy transfer (FRET). Key features of MS-qFRET include its low intrinsic background noise, high resolution and high sensitivity. This approach detects as little as 15 pg of methylated DNA in the presence of a 10,000-fold excess of unmethylated alleles, enables reduced use of PCR (8 cycles), and allows for multiplexed analyses. The high sensitivity of MS-qFRET enables one-step detection of methylation at PYCARD, CDKN2B and CDKN2A genes in patient sputum samples that contain low concentrations of methylated DNA, which normally would require a nested PCR approach. The direct application of MS-qFRET on clinical samples offers great promise for its translational use in early cancer diagnosis, prognostic assessment of tumor behavior, as well as monitoring response to therapeutic agents.

Footnotes

• • Received November 3, 2008.
  • Accepted April 28, 2009.
• Copyright © 2009, Cold Spring Harbor Laboratory Press