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

Gilbert's syndrome, due to reduced hepatic bilirubin glucuronidation is ass ociated with the presence of two extra nucleotides (TA) in the promoter reg ion of the UDP-glucuro-nosyltransferase 1 (UGT1A1) gene. A rapid method was developed to detect this genetic polymorphism, using double gradient denat uring gradient gel electrophoresis (DG-DGGE). The promoter region of the UG T1A1 gene was amplified with a 40-mer CC-clamp attached to the 5'-end of th e reverse primer. The polymerase chain reaction (PCR) product was then sepa rated by DG-DGGE using denaturant concentrations of 15-25% and polyacrylami de concentrations of 6-12%. The (TA)(6)/(TA)(6) homozygotes were clearly di stinguished from both (TA)(7)/(TA)(7) homozygotes and (TA)(6)/(TA)(7) heter ozygotes. 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 p olymorphism fast, simple, nonradioactive and suitable for a clinical routin e diagnostic laboratory, helping to establish the role of this polymorphism in individuals with jaundice due to multiple causes.