therascreen MGMT Pyro Kit

The therascreen MGMT Pyro Kit is used for quantitative measurement of methylation of four CpG sites in exon 1 of the human MGMT gene. The kit contains primers and reagents for amplification of the genes, plus buffers, primers, and reagents for quantitative methylation detection in real time using Pyrosequencing technology on the PyroMark Q24 system.

The therascreen MGMT Pyro Kit is used for quantitative measurement of methylation in four CpG sites in exon 1 of the human MGMT gene (genomic sequence on chromosome 10 from 131,265,519 to 131,265,537: CGACGCCCGCAGGTCCTCG). The MGMT (O(6)-methylguanine-DNA methyltransferase) gene encodes for an enzyme which facilitates the repair of alkylated DNA by removing alkyl groups from the O6 position of guanine. Increased methylation of the MGMT gene causes reduced expression of the gene and diminished DNA repair activity. MGMT is methylated to 25–50% in numerous cancers including brain, colon, lung, and breast.

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.

The therascreen MGMT Pyro Kit enables quantitative measurement of methylation of four CpG sites in exon 1 of the human MGMT gene (see figures " Pyrogram trace obtained after analysis of unmethylated bisulfite converted DNA from a human glioblastoma sample", " Pyrogram trace obtained after analysis of methylated bisulfite converted DNA from a human glioblastoma sample", and " Pyrogram trace obtained after analysis of highly methylated bisulfite converted control DNA") and is applicable to several fields of clinical applications, such as diagnostic evaluation, clinical research, and pharma/biopharma testing.