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The human cytochrome P450 1A2 (CYP1A2) gene produces an aryl hydrocarbon hydroxylase involved in the metabolism of endogenous compounds and xenobiotics. This enzyme is classified as polypeptide 2, subfamily A, family 1, superfamily cytochrome P450. The wild type CYP1A2*1A isoenzyme (GenBank accession number NP0007752) contains 515 amino acid residues. The CYP1A2 gene is located on the complementary strand of chromosome 15q22 and is transcribed into a wild type mRNA (GenBank accession number NM000761) containing seven exons. The exonic positions within a full-length gene reference sequence can be found in Table 1. An 11666 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 (1A2_FULL_REF) for alignment of sequence data and for reporting the positions of genetic changes.
The DMG Diversity Navigator™ for CYP1A2 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 26 sequence reactions needs to be performed on each DNA sample in order to sequence all seven exons (Figure 1). Bidirectional sequence coverage can be obtained for 6.0 kb of amplified sequence. Due to its large size, exon 2 is amplified as a whole and then sequenced in two overlapping segments; the upstream segment is designated 2A and the downstream segment is designated 2B. Exons 3, 4, and 5 are amplified as one fragment and then sequenced separately. Exon 7, another large exon, is amplified as two overlapping fragments producing an upstream amplicon designated 7A-D and a downstream amplicon designated 7B-F. Amplicon 7A-D is sequenced in three overlapping segments; the upstream segment is designated 7A, the internal segment is designated 7B, and the downstream segment is designated 7C. Amplicon 7B-F is also sequenced in three overlapping segments. The upstream segment is designated 7D, the internal segment is designated 7E, and the downstream segment is designated 7F. For the upstream fragment of exon 7 (7A-D), it may be necessary to perform duplicate genomic amplifications on each sample in order to obtain sufficient sequencing template.
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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