|
The human cytochrome P450 3A5 (CYP3A5) gene produces a niphedipine oxidase involved in the metabolism of endogenous compounds and xenobiotics. This enzyme is classified as polypeptide 5, subfamily A, family 3, superfamily cytochrome P450. The wild type CYP3A5*1A isoenzyme (GenBank accession number NP000768) contains 502 amino acid residues. The CYP3A5 gene is located on the complementary strand of chromosome 7q21 and is transcribed into a wild type mRNA (GenBank accession number NM000777) containing thirteen exons. The exonic positions within a full-length gene reference sequence can be found in Table 1. A 47695 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 (3A5_FULL_REF) for alignment of sequence data and for reporting the positions of genetic changes.
The DMG Diversity Navigator™ for CYP3A5 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 thirteen wild type exons, the system for CYP3A5 includes coverage of four variant exons which contain wild type exons 2, 9, 10 and 13 within their boundaries and three additional variants. Variant exon 4 is located in the sequence between wild type exons 3 and 4, variant exon 6 is located between wild type exons 4 and 5, and variant exon 8 is located between wild type exons 5 and 6 (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 36 sequence reactions needs to be performed on each DNA sample in order to sequence all sixteen exons (Figure 1). Bidirectional sequence coverage can be obtained for 8.7 kb of amplified sequence. Exons 4, 5, and 6 are amplified as one fragment, due to their small size and close proximity, and then exons 4 and 5 are sequenced together while exon 6 is sequenced separately. Likewise, exons 7, 8, and 9 are amplified as one fragment, and then exon 7 is sequenced separately while exons 8 and 9 are sequenced together. Exons 11 and 12 are amplified as one fragment and then exon 11 is sequenced separately while exon 12 is sequenced in two overlapping segments; the upstream segment is designated 12A and the downstream segment is designated 12B. Exon 13 is amplified as a whole and then sequenced in four overlapping segments; the outer upstream segment is designated 13A, the internal upstream segment is designated 13B, the internal downstream segment is designated 13C, and the outer downstream segment is designated 13D. If only the wild type exons are to be analyzed, there is no need to sequence exon 6, the upstream segment of exon 12 (12A), or the last three segments of exon 13 (13B, 13C, and 13D).
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.
|
