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The human cytochrome P450 2C9 (CYP2C9) gene produces a mephenytoin 4-hydroxylase involved in the metabolism of endogenous compounds and xenobiotics. This enzyme is classified as polypeptide 9, subfamily C, family 2, superfamily cytochrome P450. The wild type CYP2C9*1 isoenzyme (GenBank accession number NP000762) contains 490 amino acid residues. The CYP2C9 gene is located on chromosome 10q24 and is transcribed into a wild type mRNA (GenBank accession number M61857) containing nine exons. The exonic positions within a full-length gene reference sequence can be found in Table 1. A 76066 base pair genomic sequence from the Golden Path (www.genome.ucsc.edu) containing the entire gene and several kilobases of intergenic sequence serves as a full-length gene reference (2C9_FULL_REF) for alignment of sequence data and for reporting the positions of genetic changes.
The DMG Diversity Navigator™ for CYP2C9 enables the detection of virtually all relevant genetic variants by providing bidirectional sequence coverage for the exons and flanking intronic regions. It was not designed to screen the promoter region. As well as the nine wild type exons, the system for CYP2C9 includes coverage of five variant exons which contain wild type exons 1, 2, 3, 5, and 9 within their boundaries and four additional variants. Variant exons 2 and 3 are located in the sequence between wild type exons 1 and 2, and variant exons 12 and 13 are located after wild type exon 9 (Table 1).
A product profile is provided which includes concise protocols for every step of the experimental process. A total of 11 genomic amplifications and 32 sequence reactions needs to be performed on each DNA sample in order to sequence all thirteen exons (Figure 1). Bidirectional sequence coverage can be obtained for 8.4 kb of amplified sequence. Due to their small size and close proximity, exons 2, 3, and 4 are amplified as one fragment, and then sequenced separately. Exons 5, 11, and 12 are each amplified as a fragment, and then each fragment is sequenced in two overlapping segments designated as 5A and B, 11A and B, and 12A and B respectively.
Positions of amplicons and bidirectional sequence reads within the full-length gene reference can be found in Table 1. Sequence reads are easily assembled into contigs by alignment to the amplicon reference fragments to identify and edit sites of genetic polymorphism in the experimental sequence data. Potential polymorphic sites in experimental data may also be identified by positional information reported in Table 2. This table integrates publicly available polymorphism data with that obtained at BioVentures, Inc. and reports the variable sites by position on the full-length gene reference sequence as well as on the appropriate amplicon reference fragment.
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