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

• We developed a new plasmid reference material for UGT1A1(TA)n genotyping.
• Plasmid reference material contains all four different alleles known to be present in the human population.
• Plasmid reference material contains enough sequence upstream and downstream of polymorphic site for novel method development.
• A new fragment analysis method for UGT1A1(TA)n genotyping was developed.

BackgroundGilbert'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.MethodsPlasmids 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.ResultsPlasmids 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.ConclusionsPrepared 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.