Published online 7 September 2004
Nucleic Acids Research, Vol. 32 No. 16 © Oxford University Press 2004; all rights reserved
A novel real-time PCR assay for quantitative analysis of methylated alleles (QAMA): analysis of the retinoblastoma locus
Institut für Humangenetik, Hufelandstrasse 55, D-45122 Essen, Germany and 1 Retinoblastoma Genetic Screening Unit, Barts and The London NHS Trust, 2nd Floor Medical College, Turner Street, London E1 1AD, UK
* To whom correspondence should be addressed. Tel: ++49 201 723 4558; Fax: ++49 201 723 5900; Email: Michael.Zeschnigk{at}uni-essen.de
Received July 15, 2004; Revised and Accepted August 13, 2004
Altered methylation patterns have been found to play a role in developmental disorders, cancer and aging. Increasingly, changes in DNA methylation are used as molecular markers of disease. Therefore, there is a need for reliable and easy to use techniques to detect and measure DNA methylation in research and routine diagnostics. We have established a novel quantitative analysis of methylated alleles (QAMA) which is essentially a major improvement over a previous method based on real-time PCR (MethyLight). This method is based on real-time PCR on bisulfite-treated DNA. A significant advantage over conventional MethyLight is gained by the use of TaqMan probes based on minor groove binder (MGB) technology. Their improved sequence specificity facilitates relative quantification of methylated and unmethylated alleles that are simultaneously amplified in single tube. This improvement allows precise measurement of the ratio of methylated versus unmethylated alleles and cuts down potential sources of inter-assay variation. Therefore, fewer control assays are required. We have used this novel technical approach to identify hypermethylation of the CpG island located in the promoter region of the retinoblastoma (RB1) gene and found that QAMA facilitates reliable and fast measurement of the relative quantity of methylated alleles and improves handling of diagnostic methylation analysis. Moreover, the simplified reaction setup and robustness inherent to the single tube assay facilitates high-throughput methylation analysis. Because the high sequence specificity inherent to the MGB technology is widely used to discriminate single nucleotide polymorphisms, QAMA potentially can be used to discriminate the methylation status of single CpG dinucleotides.
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