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Nucleic Acids Research Advance Access originally published online on July 15, 2008
Nucleic Acids Research 2008 36(14):4689-4698; doi:10.1093/nar/gkn455
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Nucleic Acids Research, 2008, Vol. 36, No. 14 4689-4698
© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Molecular Biology

DNA methylation analysis by digital bisulfite genomic sequencing and digital MethyLight

Daniel J. Weisenberger1, Binh N. Trinh1, Mihaela Campan1, Shikhar Sharma2,3, Tiffany I. Long1, Suchitra Ananthnarayan4, Gangning Liang2, Francisco J. Esteva5, Gabriel N. Hortobagyi5, Frank McCormick4, Peter A. Jones2,3 and Peter W. Laird1,3,*

1Department of Surgery, 2Department of Urology, 3Department of Biochemistry and Molecular Biology, University of Southern California/Norris Comprehensive Cancer Center, Los Angeles, CA, 90033, 4Helen Diller Family Comprehensive Cancer Center and Cancer Research Institute, University of California, San Francisco, CA and 5Department of Breast Medical Oncology, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX, USA

*To whom correspondence should be addressed. Tel: +(323) 865 0650; Fax: +(323) 865 0158; Email: plaird{at}usc.edu

Received March 17, 2008. Revised June 24, 2008. Accepted June 26, 2008.

Alterations in cytosine-5 DNA methylation are frequently observed in most types of human cancer. Although assays utilizing PCR amplification of bisulfite-converted DNA are widely employed to analyze these DNA methylation alterations, they are generally limited in throughput capacity, detection sensitivity, and or resolution. Digital PCR, in which a DNA sample is analyzed in distributive fashion over multiple reaction chambers, allows for enumeration of discrete template DNA molecules, as well as sequestration of non-specific primer annealing templates into negative chambers, thereby increasing the signal-to-noise ratio in positive chambers. Here, we have applied digital PCR technology to bisulfite-converted DNA for single-molecule high-resolution DNA methylation analysis and for increased sensitivity DNA methylation detection. We developed digital bisulfite genomic DNA sequencing to efficiently determine single-basepair DNA methylation patterns on single-molecule DNA templates without an interim cloning step. We also developed digital MethyLight, which surpasses traditional MethyLight in detection sensitivity and quantitative accuracy for low quantities of DNA. Using digital MethyLight, we identified single-molecule, cancer-specific DNA hypermethylation events in the CpG islands of RUNX3, CLDN5 and FOXE1 present in plasma samples from breast cancer patients.

The authors wish it to be known that, in their opinion, the first three authors should be regarded as joint First Authors.


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[Abstract] [Full Text] [PDF]


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