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

被引：137

作者：

Kristensen, Lasse S. ^{[1
,2
]}

Mikeska, Thomas ^{[1
]}

Krypuy, Michael ^{[1
]}

Dobrovic, Alexander ^{[1
,3
]}

机构：

[1] Peter MacCallum Canc Ctr, Mol Pathol Res & Dev Lab, Dept Pathol, Melbourne, Vic 8006, Australia

[2] Univ Aarhus, Inst Human Genet, DK-8000 Aarhus C, Denmark

[3] Univ Melbourne, Dept Pathol, Parkville, Vic 3010, Australia

来源：

NUCLEIC ACIDS RESEARCH | 2008年 / 36卷 / 07期

基金：

英国医学研究理事会;

关键词：

D O I：

10.1093/nar/gkn113

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

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.

引用

页数：13

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