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MGMT Pyro Kit

For quantitative measurement of methylation in the human MGMT gene
  • Quantitative methylation detection
  • Comprehensive results in real time
  • Easy interpretation of complex sequence information
  • Sequence context as built-in control of the assay
  • Flexible post-run analysis

The 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.

Cat No./ID: 970061
MGMT Pyro Kit (48)
This MGMT Pyro Kit is intended for molecular biology applications.
This product is not intended for the diagnosis, prevention, or treatment of a disease or for patient management.

0
Unmethylated DNA from a human glioblastoma sample.

Pyrogram trace of unmethylated, bisulfite-converted DNA from a human glioblastoma sample. The bar at dispensation 15 represents the control for completion of bisulfite conversion.

1
Highly methylated control DNA.

Pyrogram trace of highly methylated, bisulfite-converted control DNA. The bar at dispensation 15 represents the control for completion of bisulfite conversion.

2
Methylated DNA from a human glioblastoma sample.

Pyrogram trace of methylated, bisulfite-converted DNA from a human glioblastoma sample. The bar at dispensation 15 represents the control for completion of bisulfite conversion.

3
Illustration of the MGMT assay.
The sequence indicated is the analyzed sequence after bisulfite conversion. Y indicates the potentially methylated sites and boxes indicate the analyzed CpG sites. The asterisk indicates the site for bisulfite conversion control. FP: Forward PCR primers; RPB: Reverse PCR primers (B indicates biotinylation); Seq: Sequencing primers.
Principle
The 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) in real time using Pyrosequencing technology on the PyroMark Q24 System. The MGMT (O(6)-methylguanine-DNA methyltransferase) gene encodes for an enzyme that 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 cancers.
Procedure
Bisulfite converted genomic DNA is amplified by PCR and then sequenced through the defined region (see figure "Illustration of 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 (see figures "Unmethylated DNA from a human glioblastoma sample", "Methylated DNA from a human glioblastoma sample", and "Highly methylated control DNA") .

Applications

The 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).

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Safety Data Sheets (1)
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