MethyLight: a high-throughput assay to measure DNA methylation.
Journal: 2000/April - Nucleic Acids Research
ISSN: 1362-4962
PUBMED: 10734209
Abstract:
Cytosine-5 DNA methylation occurs in the context of CpG dinucleotides in vertebrates. Aberrant methylation of CpG islands in human tumors has been shown to cause transcriptional silencing of tumor-suppressor genes. Most methods used to analyze cytosine-5 methylation patterns require cumbersome manual techniques that employ gel electrophoresis, restriction enzyme digestion, radiolabeled dNTPs or hybridization probes. The development of high-throughput technology for the analysis of DNA methylation would significantly expand our ability to derive molecular information from clinical specimens. This study describes a high-throughput quantitative methylation assay that utilizes fluorescence-based real-time PCR (TaqMan) technology that requires no further manipulations after the PCR step. MethyLight is a highly sensitive assay, capable of detecting methylated alleles in the presence of a 10,000-fold excess of unmethylated alleles. The assay is also highly quantitative and can very accurately determine the relative prevalence of a particular pattern of DNA methylation. We show that MethyLight can distinguish between mono-allelic and bi-allelic methylation of the MLH1 mismatch repair gene in human colorectal tumor specimens. The development of this technique should considerably enhance our ability to rapidly and accurately generate epigenetic profiles of tumor samples.
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Nucleic Acids Res 28(8): e32

MethyLight: a high-throughput assay to measure DNA methylation

Department of Surgery, Department of Biochemistry and Molecular Biology and Department of Pathology, University of Southern California School of Medicine, Norris Comprehensive Cancer Center, Mail Stop # 73, Room 6418, 1441 Eastlake Avenue, Los Angeles, CA 90033, USA and Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10021-6094, USA
To whom correspondence should be addressed at: USC/Norris Cancer Center, 1441 Eastlake Avenue, Los Angeles, CA 90089-9176, USA. Tel: +1 323 865 0650; Fax: +1 323 865 0158; Email: plaird@hsc.usc.edu
Received 2000 Jan 25; Revised 2000 Mar 3; Accepted 2000 Mar 3.

Abstract

Cytosine-5 DNA methylation occurs in the context of CpG dinucleotides in vertebrates. Aberrant methylation of CpG islands in human tumors has been shown to cause transcriptional silencing of tumor-suppressor genes. Most methods used to analyze cytosine-5 methylation patterns require cumbersome manual techniques that employ gel electrophoresis, restriction enzyme digestion, radiolabeled dNTPs or hybridization probes. The development of high-throughput technology for the analysis of DNA methylation would significantly expand our ability to derive molecular information from clinical specimens. This study describes a high-throughput quantitative methylation assay that utilizes fluorescence-based real-time PCR (TaqMan®) technology that requires no further manipulations after the PCR step. MethyLight is a highly sensitive assay, capable of detecting methylated alleles in the presence of a 10 000-fold excess of unmethylated alleles. The assay is also highly quantitative and can very accurately determine the relative prevalence of a particular pattern of DNA methylation. We show that MethyLight can distinguish between mono-allelic and bi-allelic methylation of the MLH1 mismatch repair gene in human colorectal tumor specimens. The development of this technique should considerably enhance our ability to rapidly and accurately generate epigenetic profiles of tumor samples.

Abstract

ACKNOWLEDGEMENTS

We thank Dennis Salonga and Ji Min Park for help in generating cDNAs and in designing TaqMan® oligonucleotides. This work was supported by NIH/NCI grants R01 CA 71716 (P.V.D) and R01 CA 75090 (P.W.L.). P.W.L. is a founding shareholder of ORCA Biosciences, Inc., Seattle, WA, which has a commercial interest in DNA methylation research. The research described in this paper was not supported by ORCA Biosciences.

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