Document Detail


DNA base bulge vs unmatched end formation in probe-based diagnostic insertion/deletion genotyping: genotyping the UGT1A1 (TA)(n) polymorphism by real-time fluorescence PCR.
MedLine Citation:
PMID:  11106326     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
BACKGROUND: Gilbert syndrome is a clinically inconsequential entity of mild unconjugated hyperbilirubinemia caused by an A(TA)(n)TAA insertion polymorphism (UGT1A1*28) in the promoter region of the gene coding for the enzyme UDP-glucuronosyltransferase 1 (EC 2.4.1. 17; UGT1A1). Present methods for genotyping this polymorphism are laborious. METHODS: Hybridization probes were designed complementary to the wild type (TA)(6) and to alleles with (TA)(7) and (TA)(8) repeats in the promoter region. Melting points were measured in samples representing all currently known alleles with (TA)(5) to (TA)(8) repeats. Probe melting points were predicted with a thermodynamic nearest-neighbor model for Watson-Crick paired probes. The dominant secondary structures resulting from probe hybridization were predicted by thermodynamic free energy calculations. Alternatively samples were genotyped based on amplicon size resolved by high-resolution polyacrylamide gel electrophoresis. RESULTS: Only short probes (22-24 bases) could be successfully used for genotyping this locus because of the very low stability of this TA repeat. Assays based on (TA)(7) or (TA)(8) genotype-compatible hybridization probes effectively discriminated five to eight TA repeats. The consecutive use of two different detection probes was necessary for better discrimination of some heterozygous genotypes. All results were in concordance with the alternative genotyping method. Of 100 investigated Caucasians (50 males, 50 females), 9 (9%) were homozygous for the (TA)(7) allele. CONCLUSIONS: The presented method for genotyping the (TA)(n) promoter polymorphism of the UGT1A1 gene with the LightCycler has the potential to genotype all currently known (TA)(n) repeats in a single assay and is sensitive toward possible new genotypes. Our findings also show that thermodynamic calculations are of practical value for the design of hybridization probe assays for the genotyping of insertion/deletion polymorphisms.
Authors:
N von Ahsen; M Oellerich; E Schütz
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Publication Detail:
Type:  Journal Article    
Journal Detail:
Title:  Clinical chemistry     Volume:  46     ISSN:  0009-9147     ISO Abbreviation:  Clin. Chem.     Publication Date:  2000 Dec 
Date Detail:
Created Date:  2000-12-26     Completed Date:  2001-01-04     Revised Date:  2004-11-17    
Medline Journal Info:
Nlm Unique ID:  9421549     Medline TA:  Clin Chem     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  1939-45     Citation Subset:  IM    
Affiliation:
Department of Clinical Chemistry, Georg-August-University, Robert-Koch-Strasse 40, 37075 Goettingen, Germany. nahsen@gwdg.de
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MeSH Terms
Descriptor/Qualifier:
DNA Probes*
Electrophoresis, Polyacrylamide Gel
Female
Fluorescent Dyes
Genotype
Gilbert Disease / genetics
Glucuronosyltransferase / chemistry,  genetics*
Humans
Male
Mutagenesis, Insertional
Polymerase Chain Reaction / methods
Polymorphism, Genetic
Protein Structure, Secondary
Sequence Deletion
Thermodynamics
Chemical
Reg. No./Substance:
0/DNA Probes; 0/Fluorescent Dyes; EC 2.4.1.17/Glucuronosyltransferase

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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