Sensitive Melting Analysis after Real Time- Methylation Specific PCR (SMART-MSP): high-throughput and probe-free quantitative DNA methylation detection | Zendy

Lasse S. Kristensen | Zendy; Thomas Mikeska | Zendy; Michael Krypuy | Zendy; Alexander Dobrovic | Zendy

Open Access

Sensitive Melting Analysis after Real Time- Methylation Specific PCR (SMART-MSP): high-throughput and probe-free quantitative DNA methylation detection

Author(s) -

Lasse S. Kristensen,

Thomas Mikeska,

Michael Krypuy,

Alexander Dobrovic

Publication year - 2008

Publication title -

nucleic acids research

Language(s) - English

Resource type - Journals

SCImago Journal Rank - 9.008

H-Index - 537

eISSN - 1362-4954

pISSN - 0305-1048

DOI - 10.1093/nar/gkn113

Subject(s) - biology , dna methylation , methylation , dna , real time polymerase chain reaction , high resolution melt , throughput , microbiology and biotechnology , polymerase chain reaction , illumina methylation assay , computational biology , genetics , gene , gene expression , computer science , telecommunications , wireless

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 quali- tative. We describe a new probe-free quantitative methylation-specific PCR (MSP) assay that incorpo- rates 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 methyla- tion. As proof of principle, we have developed assays for the promoter regions of the CDH1, DAPK1, CDKN2A (p16INK4a) and RARB genes. We show that highly accurate quantification is possible in the range from 100% to 0.1% methylated template when 25ng 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.

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