Quantitative detection of DNA methylation states in minute amounts of DNA from body fluids.

Quantitative and reliable estimation of DNA methylation levels for multiple genomic regions pose a major challenge where starting DNA is available in very low quantity. Here we review major advances in the development of techniques for quantitative detection of DNA methylation in minute amount of DNA and describe a detailed protocol for quantitative Methylation Analysis of Minute DNA amounts after whole Bisulfitome Amplification (qMAMBA), a combination of techniques that allows quantitative and sensitive detection of DNA methylation at multiple CpG sites and for multiple gene assays. Recently we successfully used this technique to quantitatively detect DNA methylation for a set of cancer-related genes in lung cancer patient plasma samples [18]. This method involves genome-wide amplification of bisulfite-modified DNA template followed by quantitative methylation detection using pyrosequencing. This allows a precise assessment of DNA methylation at CpG sites and could be adapted for high-throughput settings. It can also be applied in conjunction with studies involving single-cells or laser capture microdissected samples. Thus, this method should facilitate DNA methylation studies aiming to discover epigenetic biomarkers, and should prove particularly valuable in profiling a large sample series of body fluids from molecular epidemiology studies.