title: MethySYBR, a novel quantitative PCR assay for the dual analysis of DNA methylation and CpG methylation density.
abstract: 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.
authors.affiliation: Breast Cancer Program, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, 1650 Orleans St., CRBI-143, Baltimore, MD 21231, USA.
authors.forename: Pang-Kuo
authors.initials: PK
authors.lastname: Lo
authors.forename: Hanano
authors.initials: H
authors.lastname: Watanabe
authors.forename: Pi-Chun
authors.initials: PC
authors.lastname: Cheng
authors.forename: Wei Wen
authors.initials: WW
authors.lastname: Teo
authors.forename: Xiaohui
authors.initials: X
authors.lastname: Liang
authors.forename: Pedram
authors.initials: P
authors.lastname: Argani
authors.forename: Ji Shin
authors.initials: JS
authors.lastname: Lee
authors.forename: Saraswati
authors.initials: S
authors.lastname: Sukumar
mesh_terms: D002460:Cell Line; D019175:DNA Methylation / Q000235:genetics*; D015226:Dinucleoside Phosphates / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results
chemical_list: D015226:Dinucleoside Phosphates; C015772:CpG dinucleotide