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The human cytochrome P450 2C19 (CYP2C19) gene produces a mephenytoin 4-hydroxylase involved in the metabolism of endogenous compounds and xenobiotics. This enzyme is classified as polypeptide 19, subfamily C, family 2, superfamily cytochrome P450. The wild type CYP2C19*1A isoenzyme (GenBank accession number NP000760) contains 490 amino acid residues. The CYP2C19 gene is located on chromosome 10q24 and is transcribed into a wild type mRNA (GenBank accession number M61854) containing nine exons. The exonic positions within a full-length gene reference sequence can be found in Table 1. A 135014 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 (2C19_FULL_REF) for alignment of sequence data and for reporting the positions of genetic changes.
The DMG Diversity Navigator™ for CYP2C19 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 CYP2C19 includes coverage of four variant exons which contain wild type exons 1, 4, and 6 within their boundaries (Table 1).
A product profile is provided which includes concise protocols for every step of the experimental process. A total of 8 genomic amplifications and 18 sequence reactions needs to be performed on each DNA sample in order to sequence all nine exons (Figure 1). Bidirectional sequence coverage can be obtained for 4.3 kb of amplified sequence. Due to their small size and close proximity, exons 2 and 3 are amplified and sequenced as one fragment. Exon 9 is amplified as a whole and then sequenced in two overlapping segments; the upstream segment is designated 9A and the downstream segment is designated 9B.
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 the 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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