Maximum data stringency in CYP450 Analysis

In the area of Cytochrome P450 (CYP450) genotyping, a number of polymorphisms with pharmacogenetic implications have already been identified, but pharmaceutical research will certainly identify different and more pharmacogenetic markers that need to be analysed and validated. This is precisely where Pyrosequencing technology adds value: Pyrosequencing technology can today provide a functional assay for any CYP450 mutation, and for any new marker in the future. Thanks to the flexibility of primer placement in Pyrosequencing assays, virtually no genetic marker is out of reach.

CYP genes that can be analysed by Pyrosequencing today

The strength of Pyrosequencing technology lies in its ability to characterise and validate a genetic marker with total reliability. Researchers studying pharmacogenetic markers, specifically CYP450 mutations in 2D6, 2C19, 2C9, 3A4 and 3A5, have found the reliability of Pyrosequencing technology in validation to be a significant benefit. Pharmaceutical companies are highly interested in our CYP450 marker assays, now that they are planning to include pharmacogenetic data with their new drug submissions. Pyrosequencing technology will provide quality-controlled, pharmacogenetic data for each individual sample analyzed, leading to superior accuracy of individual genotypes - a fact that must be very interesting to any organization that demands data stringency at the early phase of a pharmacogenetic study.

CYP alleles that can be analysed by Pyrosequencing

Polymorphisms with sequence context

• Pyrosequencing technology presents CYP mutations with the sequence context surrounding the mutation. This ensures that accurate and unambiguous results are obtained from a single run - no need to run replicates. Sequence context also acts as a built-in quality control, ensuring that the correct position has been analyzed.
  

Example of Pyrogram result, using CYP3A4, with polymorphic position A/A highlighted. Sequence reads A/GGAGAGA, the above pyrogram shows the genotype A/A.

CYP2D6 multiplex assay for the identification of alleles 2, 3 and 7. In the upper part of the figure, the PCR fragment and sequences to be identified are shown in color for the three positions. The same colors show the expected incorporation in the histograms (left) and the corresponding Pyrogram traces are presented to the right. Polymorphic positions are marked with yellow. A) Normal genotype B) Heterozygous allele 3 C) Heterozygous allele 2.    click to enlarge