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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
Author(s) -
Gürtler Volker,
Parkin John D.,
Mayall Barrie C.
Publication year - 1999
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/(sici)1522-2683(19991001)20:14<2841::aid-elps2841>3.0.co;2-v
Subject(s) - gene , temperature gradient gel electrophoresis , gel electrophoresis , microbiology and biotechnology , genetics , biology , chemistry , 16s ribosomal rna
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.