Gilbert's syndrome is the most common inherited genetic disorder of bilirubin metabolism, resulting from changes in nucleotide sequence in the uridine diphosphate-glucuronyl-transferase 1A1 gene (UGT1A1). The most common change is the insertion of one or two additional TA pairs into the A(TA)6TAA sequence located in the non-coding promoter region of exon 1A1. Gilbert’s syndrome is a benign disorder, which does not require specific treatment. However, its diagnosis is still very important, because these patients are more susceptible to side effects of certain drugs such as irinotecan. Unfortunately, there are no commercially available certified reference materials, that would be helpful in determining the number of (TA)n repeats and whose nucleotide sequence would be determined by sequencing. The goal of this thesis was to prepare reference material which would be an indispensable part of the molecular analysis of (TA)n repeat polymorphism and which could be produced in large quantities using bacteria, thus ensuring its unlimited supply. The reference material could also be used to further explore and develop even faster and more efficient methods for the determination of microsatellite lengths such as (TA)n repeat. First, the conditions for polymerase chain reaction were optimized and the segment of UGT1A1 gene containing the (TA)n repeat was amplified. Amplified segment was isolated from agarose gel, cleaved with restriction endonucleases and inserted into the plasmid vector. Plasmid constructs were transformed into competent bacterial cells and inoculated onto the selective plates. Random bacterial colonies were chosen and their plasmid DNA was isolated. We then amplified segment of plasmid DNA, which contained the polymorphism and analyzed it using fragment analysis to determine the size and therefore the number of (TA)n repeats in the plasmid. Sequence was determined for selected plasmids for each (TA)n repeat to further verify their suitability for use as a reference material. We were able to produce plasmids with 4, 5, 6, 7 and 8 (TA)n repeats, which can be assembled in any heterozygous or homozygous genotype. Reference material will serve as an internal standard, which will allow evaluation of analysis quality. At the same time it will help with the interpretation of analysis results of samples with unknown number of (TA)n repeats. Produced plasmid constructs will be used as a tool to create new analytical methods for genotyping, which will allow to discriminate rare (TA)n repeats, which until now were not available in sufficient amounts to optimize other potential genotyping methods.
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