Assaying DNA Methylation Based on High-Throughput Melting Curve Approaches

• Published in: Genomics (San Diego, Calif.) Calif.), 2002-10, Vol.80 (4), p.376-384
• Main Authors: Akey, Dayna T, Akey, Joshua M, Zhang, Kun, Jin, Li
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; DNA - analysis; DNA - chemistry; DNA - metabolism; DNA Methylation; Fundamental and applied biological sciences. Psychology; Genes, p16 - physiology; Humans; Molecular and cellular biology; Molecular genetics; Mutagenesis. Repair; Polymerase Chain Reaction; Temperature; Tumor Cells, Cultured
• ISSN: 0888-7543; 1089-8646
• DOI: 10.1006/geno.2002.6851

Here we describe two high-throughput methods to assay DNA methylation, melting curve methylation specific PCR (McMSP) and melting curve combined bisulfite restriction analysis (McCOBRA), which adapt standard MSP and COBRA methods to a melting curve analysis based platform. We show that McMSP and McCOBRA can accurately determine methylation status in a high-throughput and gel-free manner. Moreover, McCOBRA can be used to quantitatively estimate the percent of methylated DNA at a specific CpG site within a heterogeneous sample. The accuracy of McMSP and McCOBRA was initially tested using the 5′-CpG site of the tumor-suppressor gene CDKN2A as a model system in homogeneous and heterogeneous controls, and cancer cell line samples. Furthermore, the robustness of McMSP and McCOBRA was validated in four additional loci. We demonstrate that McCOBRA and McMSP provide several advantages over existing methods, as they are simple, accurate, and high-throughput, which makes them widely applicable to large-scale methylation studies.