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Methods

Multiplexed profiling of candidate genes for CpG island methylation status using a flexible PCR/LDR/Universal Array assay

¹ Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York 10021, USA ² Department of OB/GYN, Columbia University Medical Center, New York, New York 10032, USA ³ Departments of Pediatrics and Molecular Genetics, University of Medicine and Dentistry of New Jersey (UMDNJ)-Robert Wood Johnson Medical School, New Brunswick, New Jersey 08901, USA ⁴ Department of Surgery, Colorectal Surgery Service, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA

DNA methylation in CpG islands is associated with transcriptional silencing. Accurate determination of cytosine methylation status in promoter CpG dinucleotides may provide diagnostic and prognostic value for human cancers. We have developed a quantitative PCR/LDR/Universal Array assay that allows parallel evaluation of methylation status of 75 CpG dinucleotides in the promoter regions of 15 tumor suppressor genes (CDKN2B, CDKN2A, CDKN2D, CDKN1A, CDKN1B, TP53, BRCA1, TIMP3, APC, RASSF1, CDH1, MGMT, DAPK1, GSTP1, and RARB). When compared with an independent pyrosequencing method at a single promoter, the two approaches gave good correlation. In a study using 15 promoter regions and seven blinded tumor cell lines, our technology was capable of distinguishing methylation profiles that identified cancer cell lines derived from the same origins. Preliminary studies using 96 colorectal tumor samples and 73 matched normal tissues indicated CpG methylation is a gene-specific and nonrandom event in colon cancer. This new approach is suitable for clinical applications where sample quantity and purity can be limiting factors.

⁵ Corresponding author.
E-mail barany{at}med.cornell.edu; fax (212) 746-8104.

[Supplemental material is available online at www.genome.org.]

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