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.
University of Ljubljana, Faculty of Pharmacy, Department of Clinical Biochemistry, Askerceva cesta 7, SI-1000 Ljubljana, Slovenia.
Vid
V
Mlakar
University of Ljubljana, Faculty of Pharmacy, Department of Clinical Biochemistry, Askerceva cesta 7, SI-1000 Ljubljana, Slovenia.
Simona Jurković
SJ
Mlakar
University of Ljubljana, Faculty of Pharmacy, Department of Clinical Biochemistry, Askerceva cesta 7, SI-1000 Ljubljana, Slovenia.
Janja
J
Marc
University of Ljubljana, Faculty of Pharmacy, Department of Clinical Biochemistry, Askerceva cesta 7, SI-1000 Ljubljana, Slovenia. Electronic address: barbara.ostanek@ffa.uni-lj.si.
Barbara
B
Ostanek
D000483:Alleles; D002851:Chromatography, High Pressure Liquid; D060005:Genotyping Techniques / Q000592:standards*; D005878:Gilbert Disease / Q000235:genetics; D014453:Glucuronosyltransferase / Q000235:genetics*; D006801:Humans; D011110:Polymorphism, Genetic; D012015:Reference Standards; D017422:Sequence Analysis, DNA; D000099148:UGT1A1 Enzyme
D014453:Glucuronosyltransferase; D000099148:UGT1A1 Enzyme
Cloning; Fragment analysis; Reference material; Sequencing; UGT1A1