|
The human cytochrome P450 2B6 (CYP2B6) gene produces a phenobarbital-inducible protein involved in the metabolism of endogenous compounds and xenobiotics. This enzyme is classified as polypeptide 6, subfamily B, family 2, superfamily cytochrome P450. The wild type CYP2B6*1A isoenzyme (GenBank accession number NP000758) contains 491 amino acid residues. The CYP2B6 gene is located on chromosome 19q13 and is transcribed into a wild type mRNA (GenBank accession number NM000767) containing nine exons. The exonic positions within a full-length gene reference sequence can be found in Table 1. A 31099 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 (2B6_FULL_REF) for alignment of sequence data and for reporting the positions of genetic changes.
The DMG Diversity Navigator™ for CYP2B6 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 24 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 5.5 kb of amplified sequence. Due to their small size and close proximity, exons 2 and 3 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 four overlapping segments; the outer upstream segment is designated 9A, the internal upstream segment is designated 9B, the internal downstream segment is designated 9C, and the outer downstream segment is designated 9D. For exon 9, 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.
|
