Preparation of reference material for UGT1A1 (TA)n polymorphism genotyping

Background: Gilbert's syndrome is one of the most common metabolic syndromes in the human population characterised by mild unconjugated hyperbilirubinemia resulting from reduced activity of the bilirubin conjugating enzyme UDP-glucuronosyltransferase (UGT1A1). Although Gilbert's syndrome is usually quite benign UGT1A1(TA)(n) genotyping is important in exclusion of more serious causes of hyperbilirubinemia and since it has significant implications for personalised medicine. The aim of our study was to develop plasmid based reference materials which could be used for UGT1A1(TA)(n) genotyping. Methods: Plasmids were generated using recombinant DNA technology and their number of repeats as well as the entire sequence verified by Sanger sequencing. Their suitability as reference materials was tested using sizing by capillary electrophoresis and denaturing high performance liquid chromatography. Results: Plasmids containing all four different alleles (TA)(5), (TA)(6), (TA)(7) and (TA)(8) that are present in the human population as well as a plasmid with (TA)(4) repeats were successfully generated. Conclusions: Prepared plasmid reference materials allow the creation of all possible UGT1A1(TA)(n) polymorphism genotypes and can serve as an efficient substitute for the human genomic DNA reference material in routine genotyping and in the development of new genotyping tests. (C) 2014 Elsevier B.V. All rights reserved.