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The human P450 oxidoreductase (POR) gene produces a NADPH reductase which is the electron donor for the cytochrome P450 superfamily of monooxygenases. The wild type POR isoenzyme (GenBank accession number NP000932) contains 680 amino acid residues. The POR gene is located on chromosome 7q11 and is transcribed into a wild type mRNA (GenBank accession number NM000941) containing fifteen exons. The exonic positions within a full-length gene reference sequence can be found in Table 1. A 49311 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 (POR_FULL_REF) for alignment of sequence data and for reporting the positions of genetic changes.
The DMG Diversity Navigator™ for POR 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 7 genomic amplifications and 24 sequence reactions needs to be performed on each DNA sample in order to sequence all fifteen exons (Figure 1). Bidirectional sequence coverage can be obtained for 6.0 kb of amplified sequence. Due to their small size and close proximity, exons 4, 5, 6 and 7 are amplified as one fragment; then exons 4 and 7 are sequenced separately while exons 5 and 6 are sequenced as one segment. Exons 8 and 9 are amplified and sequenced together. Exons 10 through 15 are amplified as two overlapping fragments, exons 10 through 14 and exons 12 through 15. Exons 10 and 11 are sequenced as one segment, exon 12 is sequenced separately, exons 13 and 14 are sequenced as one segment, and exon 15 is sequenced in two overlapping segments; the upstream segment is designated 15A and the downstream segment is designated 15B. For exons 4 through 6, 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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