Bisulfite converted genomic DNA is amplified by PCR and then sequenced through the defined region (see figure "
Illustration of the therascreen MGMT assay"). Sequences surrounding the defined positions serve as normalization and reference peaks for quantification and quality assessment of the analysis.
After PCR using primers targeting the defined region of exon 1, the amplicons are immobilized on Streptavidin Sepharose High Performance beads. Single-stranded DNA is prepared, and the sequencing primers anneal to the DNA. The samples are then analyzed on the PyroMark Q24 system.