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The human cytochrome P450 2A6 (CYP2A6) gene produces a coumarin
7-hydroxylase involved in the metabolism of endogenous compounds and
xenobiotics. This enzyme is classified as polypeptide 6, subfamily A,
family 2, superfamily cytochrome P450. The wild type CYP2A6*1A isoenzyme (GenBank accession number NP000753) contains 494 amino acid residues. The CYP2A6 gene is located on the complementary strand of chromosome 19q13 and is transcribed into a wild type mRNA (GenBank accession number NM000762) containing nine exons. The exonic positions within a full-length gene reference sequence can be found in Table 1. A 10898 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 (2A6_FULL_REF) for alignment of sequence data and for reporting the positions of genetic changes.
The DMG Diversity Navigator™ for CYP2A6 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, nor was it designed to screen variant exons.
A product profile is provided which includes concise protocols for every step of the experimental process. A total of 6 genomic amplifications and 20 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 3 and 4 are amplified as one fragment and then sequenced separately. Exons 5 and 6 are treated in the same manner, as are exons 7 and 8. 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 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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