Use of double gradient denaturing gradient gel electrophoresis to detect (AT)n polymorphisms in the UDP-glucuronosyltransferase 1 gene promoter associated with Gilbert's syndrome. |
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Authors: | V Gürtler J D Parkin B C Mayall |
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Affiliation: | Division of Laboratory Medicine, Austin & Repatriation Medical Centre, Heidelberg, Victoria, Australia. volker@austin.unimelb.edu.au |
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Abstract: | Gilbert's syndrome, due to reduced hepatic bilirubin glucuronidation is associated with the presence of two extra nucleotides (TA) in the promoter region of the UDP-glucuronosyltransferase 1 (UGT1A1) gene. A rapid method was developed to detect this genetic polymorphism, using double gradient denaturing gradient gel electrophoresis (DG-DGGE). The promoter region of the UGT1A1 gene was amplified with a 40-mer GC-clamp attached to the 5'-end of the reverse primer. The polymerase chain reaction (PCR) product was then separated by DG-DGGE using denaturant concentrations of 15-25% and polyacrylamide concentrations of 6-12%. The (TA)6/(TA)6 homozygotes were clearly distinguished from both (TA)7/(TA)7 homozygotes and (TA)6/(TA)7 heterozygotes. The (TA)7 allele frequency was consistent with that previously reported and elevated bilirubin levels correlated with the presence of the (TA)7 allele. The DG-DGGE method described will make detection for this polymorphism fast, simple, nonradioactive and suitable for a clinical routine diagnostic laboratory, helping to establish the role of this polymorphism in individuals with jaundice due to multiple causes. |
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