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The human cytochrome P450 2C8 (CYP2C8) gene produces a mephenytoin 4-hydroxylase involved in the metabolism of endogenous compounds and xenobiotics. This enzyme is classified as polypeptide 8, subfamily C, family 2, superfamily cytochrome P450. The wild type CYP2C8*1A isoenzyme (GenBank accession number NP000761) contains 490 amino acid residues. The CYP2C8 gene is located on the complementary strand of chromosome 10q23 and is transcribed into a wild type mRNA (GenBank accession number NM000770) containing nine exons. The exonic positions within a full-length gene reference sequence can be found in Table 1. A 34999 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 (2C8_FULL_REF) for alignment of sequence data and for reporting the positions of genetic changes.
The DMG Diversity Navigator&trade for CYP2C8 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. The system for CYP2C8 includes coverage of a variant exon which is located between wild type exons 7 and 8 (Table 1).
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 10 exons (Figure 1). Primer pairs for seven optional nested amplifications are provided in the event that the genomic amplifications lack the specificity or yield necessary to serve as sequencing template. The nested amplification primers for the CYP2C8 system may also be used to amplify directly from genomic DNA. It may be advisable to amplify exons 7 and 8 individually using the nested amplification primers rather than together as one region since this is the weakest of the three multiple-exon genomic amplification for CYP2C8.
Bidirectional sequence coverage can be obtained for 4.5 kb of amplified sequence. Exons 1, 2, and 3 are amplified together from genomic DNA as one fragment. If yields are low, this amplicon is subjected to three nested amplifications producing 3 smaller fragments, each containing one exon. Exons 7 and 8 are treated in the same manner, as are exons 9 and 10.
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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