Journal: Pharmacogenetics and genomics
Article Title: CYP2D6: novel genomic structures and alleles
Figure Lengend Snippet: PCR fragment analysis. The PCR products for amplicons F–J were used in fragment analysis on the Agilent Technologies DNA 7500 Kit to show the presence or absence of the CYP2D7 gene, CYP2D6 genes, or recombinant allele. Fragments A–E are not shown as these were primarily useful for the generation of amplicons used in allele-specific primer extension assay reactions and sequencing reactions. Genotypes tested are shown at the top of each lane. The ladder in the left column shows the size in base pairs of the molecular weight marker. The uppermost purple band in all sample lanes represents a marker depicting the maximum size that the kit can detect reliably. (a) PCR fragment F analysis detects CYP2D7 exon 9 through the region 3′ of rep 6. The * 68 +* 4-like and * 4N +* 4-like arrangements did not produce a product because the * 4-like allele did not have the exon 9 conversion present. The sample containing the * 5 /* 5 genotype and those with single * 36 , * 61 , and * 63 alleles generated an amplicon. The size of the product is consistent with the presence of the CYP2D7 -derived spacer sequence which was confirmed by DNA sequencing. (b) PCR fragment G analysis that detects CYP2D7/CYP2D6 genes. Only samples with * 13 and * 16 generated an amplicon. (c) PCR fragment H analysis detects DNA spanning from the CYP2D7 promoter to CYP2D6 promoter regions. The presence or absence of 1.6 kb spacer sequence can be visualized based on the amplicon size. Genotype * 5 /* 5 lacks the CYP2D6 promoter region, so does not generate an amplicon. Genotype * 13 +* 2A /* 5 lacks the 1.6 kb spacer sequence, so the amplicon is smaller than the products generated by the other genotypes tested. (d) PCR fragment I analysis generates an amplicon spanning from CYP2D8 3′region to a unique region in CYP2D6 intron 3. The * 13 +* 2A -containing sample generated an amplicon because CYP2D7 was absent. No amplicon was detected in * 16 samples because the crossover region in * 16 occurs after exon 7, which eliminates the reverse primer binding site for the amplicons. Normal gene arrangements and typical duplication arrangements do not generate a product because the size of the amplicons is too great. (e) PCR fragment J analysis generates amplicons spanning from the CYP2D8 3′ region to the CYP2D6 -specific trailing sequences. Amplicons are generated for the * 13 +* 2A -containing and * 16 -containing samples because CYP2D7 is absent in these samples. Normal gene arrangements and typical duplication arrangements do not generate a product because the size of the amplicons is too great.
Article Snippet: The gene copy number was determined on samples with duplication signals by the CYP2D6 ASPE kit by using the TaqMan CYP-2D6 Gene Copy Number Assay (Applied Biosystems, Foster City, California, USA, Assay ID Hs00010001_cn) per product literature.
Techniques: Polymerase Chain Reaction, Recombinant, Primer Extension Assay, Sequencing, Molecular Weight, Marker, Generated, Amplification, Derivative Assay, DNA Sequencing, Binding Assay