title: Snapback primer genotyping of the Gilbert syndrome UGT1A1 (TA)(n) promoter polymorphism by high-resolution melting.
abstract: BACKGROUND
  Gilbert syndrome, a chronic nonhemolytic unconjugated hyperbilirubinemia, is associated with thymine-adenine (TA) insertions in the UGT1A1 (UDP glucuronosyltransferase 1 family, polypeptide A1) promoter. The UGT1A1 promoter genotype also correlates with toxicity induced by the chemotherapeutic drug irinotecan. Current closed-tube assays for genotyping the UGT1A1 (TA)(n) promoter polymorphism require multiple labeled probes and/or have difficulty classifying the (TA)(5) and (TA)(8) alleles.
  
  
  METHODS
  An unlabeled 5' extension on one primer that creates a hairpin after asymmetric PCR was used to develop a snapback primer high-resolution melting assay for the (TA)(n) polymorphism. A new method that plots the local deviation from exponential decay to improve genotype clustering was used to remove background fluorescence and to analyze the data. The snapback assay was compared with small-amplicon melting and fragment length analyses in a blinded study of DNA samples from 100 African Americans.
  
  
  RESULTS
  Genotyping results obtained by small-amplicon melting and snapback primer melting were 83% and 99% concordant, respectively, with results obtained by fragment analysis. Reanalysis of the single discordant sample in the results of the snapback genotyping assay and the fragment analysis revealed an error in the fragment analysis. High-resolution melting was required for accurate snapback genotyping of the UGT1A1 (TA)(n) polymorphism. The 100% accuracy obtained with a capillary-based instrument fell to ≤81% with plate-based instruments.
  
  
  CONCLUSIONS
  In contrast to small-amplicon genotyping, snapback primer genotyping can distinguish all UGT1A1 promoter genotypes. Rapid-cycle PCR combined with snapback primer analysis with only 2 unlabeled PCR primers (one with a 5' extension) and a saturating DNA dye can genotype loci with several alleles in <30 min.
authors.affiliation: Department of Pathology, University of Utah Health Sciences Center, Salt Lake City, UT 84132, USA.
authors.forename: Jared S
authors.initials: JS
authors.lastname: Farrar
authors.forename: Robert A
authors.initials: RA
authors.lastname: Palais
authors.forename: Carl T
authors.initials: CT
authors.lastname: Wittwer
mesh_terms: D001741:Black or African American; D017931:DNA Primers; D005838:Genotype; D005878:Gilbert Disease / Q000235:genetics*; D014453:Glucuronosyltransferase / Q000235:genetics*; D006801:Humans; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D016385:TATA Box; D044366:Transition Temperature; D000099148:UGT1A1 Enzyme
chemical_list: D017931:DNA Primers; D014453:Glucuronosyltransferase; D000099148:UGT1A1 Enzyme