MethylMeter® is a sensitive MSRE-free and bisulfite-free DNA methylation detection technology for the quantitative measurement of the methylation level of specific DNA targets, even in challenging clinical samples, including FFPE tissue and bodily fluids.
Detailed procedures for the use of MethlyMeter are availabe here, in the June 2016 Journal of Epigenomics.
MethylMeter uses Ribomed’s proprietary paramagnetic bead bound Me-CpG-binding domain (MBD) protein, MethylMagnet, to separate methylated DNA from unmethylated DNA. [Step 1]. MethylMagnet takes advantage of the high specificity of the MBD2 methyl-CpG binding domain and its bias for clustered CpGs, which favors the analysis of CpG islands. MethylMeter’s detection sensitivity is achieved with a novel signal generation process called Abscription® (Abortive Transcription). Abscription involves the reiterative synthesis of short ‘abortive’ RNA transcripts by a proprietary RNA polymerase (Abscriptase®) that remains bound at an Abortive Promoter Cassette (APC) generating multiple copies of a specific, short Abortive transcript (Abscript).
After separation with MethylMagnet Kits, APCs are attached to the DNA in the methylated and unmethylated fractions in a process call CAPS (Coupled Abscription PCR Signaling), [Step 2]. The APCs are attached to the amplicons generated from the DNA targets in the methylated and unmethylated fractions, and the amount of each abscript produced is a measure of the amount of the original DNA target in each fraction and can be used to determine the percentage methylation for that target.
In the fluorescent MethylMeter assays (Format II), each Abscript binds to a quenched molecular beacon and is then extended with CAPS polymerase in a beacon-dependent reaction to convert the single-stranded hairpin form of the beacon to the double stranded form. This separates the fluorophore from the quencher on the beacon and generates a fluorescent signal. The signal amplification provided by Abscription allows detection of as little as 6 copies of DNA in 36 cycles without preamplification or nested PCR, reducing the nonspecific amplification associated with higher cycle numbers.