Sensitive Melting Analysis after Real Time- Methylation Specific PCR (SMART-MSP): high-throughput and probe-free quantitative DNA methylation detection.
DNA methylation changes that are recurrent in cancer have generated great interest as potential biomarkers for the early detection and monitoring of cancer. In such situations, essential information is missed if the methylation detection is purely qualitative. We describe a new probe-free quantitative methylation-specific PCR (MSP) assay that incorporates evaluation of the amplicon by high-resolution melting (HRM) analysis. Depending on amplicon design, different types of information can be obtained from the HRM analysis. Much of this information cannot be obtained by electrophoretic analysis. In particular, identification of false positives due to incomplete bisulphite conversion or false priming is possible. Heterogeneous methylation can also be distinguished from homogeneous methylation. As proof of principle, we have developed assays for the promoter regions of the CDH1, DAPK1, CDKN2A (p16(INK4a)) and RARB genes. We show that highly accurate quantification is possible in the range from 100% to 0.1% methylated template when 25 ng of bisulphite-modified DNA is used as a template for PCR. We have named this new approach to quantitative methylation detection, Sensitive Melting Analysis after Real Time (SMART)-MSP.
Kristensen
Lasse S
LS
Molecular Pathology Research and Development Laboratory, Department of Pathology, Peter MacCallum Cancer Centre, Locked Bag 1 A'eckett Street, Melbourne, Victoria 8006, Australia.
Mikeska
Thomas
T
Krypuy
Michael
M
Dobrovic
Alexander
A
D051017:Apoptosis Regulatory Proteins / Q000235:genetics; D001943:Breast Neoplasms / Q000235:genetics; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000235:genetics; D045744:Cell Line, Tumor; D019175:DNA Methylation; D004273:DNA, Neoplasm / Q000737:chemistry*; D064550:Death-Associated Protein Kinases; D005456:Fluorescent Dyes; D016147:Genes, Tumor Suppressor; D019942:Genes, p16; D006801:Humans; D009369:Neoplasms / Q000175:diagnosis*; D009691:Nucleic Acid Denaturation; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000379:methods*
D051017:Apoptosis Regulatory Proteins; D004273:DNA, Neoplasm; D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; C579096:DAPK1 protein, human; D064550:Death-Associated Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases