SNP/Mutation Analysis

• Analyze single and multiple SNPs and insertion/deletion mutations within 200bp stretches of the genome.

• Pyrosequencing technology presents SNPs in the context of the surrounding sequence. This guarantees that each individual analysis is correct, without the need for other controls. In a clinical context, this level of certainty is highly valuable, especially as the genetic methods developed in today’s research mature into tomorrow’s genetic tests. 

• Results from mutation analysis of codons 12 and 13 using PyroMark KRAS v2.0 test and PyroMark Q24. The upper Pyrogram trace shows a sample with a normal genotype. The middle Pyrogram trace shows mutation analysis in a sample with a G to A mutation in position 2 of codon 12, and the lower Pyrogram trace shows a sample with a G to T mutation in position 1 of codon 12 identified post-run by the altered sequence. Light blue areas indicate the variable position. (From Qiagen)

DNA Methylation Analysis

• Quantitatively analyze PCR products amplified from bisulphite treated genomic DNA for levels of methylation at single or multiple CpG sites across the genome.

Typical workflow for analysis of CpG methylation
• The approach to analyze methylation exploits one of the most powerful attributes of Pyrosequencing, namely its readout (the peaks in the Pyrogram). Unlike dideoxy (Sanger) sequencing, the peak heights produced by Pyrosequencing quantitatively report the proportions of alleles.

• Pyro Q-CpG quantifies methylation in explicit sequence context, and is both fast and easy to perform. Assay design is flexible, since the distance from the first base to be sequenced can be varied, and therefore the primer can usually be positioned in a region free of CpG sites. In addition, there are four options for design; the assay can be performed in forward or reverse orientations on either the top or the bottom strands.

• Unmethylated C (red) and methylated C (green) are differentiated by bisulfite treatment and PCR. The ratio C:mC at each CpG site (peaks in yellow column) is measured in sequence context. C not followed by G acts as control for the bisulfite step (blue column). • Sequence context is an important control since bisulfite-treated, PCR-amplified DNA is AT-rich, which decreases sequence variation. Pyro Q-CpG therefore guarantees that the correct sequence was analysed. • Pyro Q-CpG assays can contain an internal control for bisulfite treatment. C that is not followed by G in sequence is not methylated, and should therefore be fully converted to T by bisulfite and PCR. To confirm this, all templates should show only T and zero C in this position (blue column). • Pyro Q-CpG is practical in terms of starting material and throughput. DNA is readily analyzed from both fresh frozen tissue as well as the short PCR fragment sizes that are typical of paraffin-embedded tissue, where restriction fragment analysis would be difficult. (Source: Qiagen)