JMD IPSOGEN Blood Cancer Molecular Assays
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Journal of Molecular Diagnostics 2009, Vol. 11, No. 5
Copyright © 2009 American Society for Investigative Pathology & Association for Molecular Pathology
DOI: 10.2353/jmoldx.2009.080126

Technical Advances

MethySYBR, a Novel Quantitative PCR Assay for the Dual Analysis of DNA Methylation and CpG Methylation Density

Pang-Kuo Lo*, Hanano Watanabe*{dagger}, Pi-Chun Cheng*{dagger}, Wei Wen Teo*, Xiaohui Liang*, Pedram Argani{ddagger}, Ji Shin Lee§ and Saraswati Sukumar*

From the Breast Cancer Program, Department of Oncology, * and the Department of Pathology, {ddagger} Johns Hopkins University School of Medicine, Baltimore, Maryland; the Molecular and Cellular Biology Program, Biology Department, {dagger} Johns Hopkins University, Baltimore, Maryland; and the Department of Pathology, § Chonnam National University Medical School and Research Institute of Medical Sciences, Gwangju, Republic of Korea

Development of facile, sensitive, specific, and economical assays for the analysis of methylated alleles is crucial to the use of methylated biomarkers for cancer detection. We hereby report a novel method, MethySYBR, a SYBR green-based PCR assay for the dual analysis of DNA methylation and CpG methylation density. MethySYBR begins with multiplex PCR to enable the simultaneous amplification of many discrete target alleles in a single reaction using as little as 3 pg of bisulfite-converted DNA. In the second round of PCR, the specific methylated target is quantified from multiplex products using both nested methylation-independent and methylation-specific primer sets. Moreover, the use of SYBR green dye during quantitative PCR enables melting curve analysis of target amplicons to determine the methylation density of CpG sites on target alleles. To establish proof of principle, two cancer-specific methylated genes, RASSF1A and OGDHL, were assessed by MethySYBR. We demonstrated that MethySYBR sensitively detected methylated alleles in the presence of a 100,000-fold excess of unmethylated allele. Furthermore, MethySYBR was shown to be capable of analyzing minute amounts of DNA from paraffin-embedded tissue. Therefore, the MethySYBR assay is a simple, highly specific, highly sensitive, high-throughput, and cost-effective method that is widely applicable to basic and clinical studies of DNA methylation.






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Copyright © 2009 by the American Society for Investigative Pathology and the Association for Molecular Pathology.