Background: Polymorphisms in HSD11B1, the gene encoding 11?-hydroxysteroid dehydrogenase type 1 enzyme, have been associated with obesity, metabolic syndrome, and type 2 diabetes. In this study, we present an optimized high-resolution melting (HRM) method for genotyping two common polymorphisms of the HSD11B1 gene: rs846910: G)A and rs45487298: insA. Methods: One hundred DNA samples from patients with polycystic ovary syndrome and healthy controls were genotyped by HRM. The results were compared with those obtained with classic polymerase chain reaction followed by restriction fragment length polymorphism analysis. Results: Various approaches were used during HRM specificity optimization. With the optimized method, genotyping accuracy of 100% was achieved. Conclusions: HRM analysis is a fast, simple, and cost-effective method compared with the alternative genotyping approaches. The work required for optimizing the method (improvement of specificity) is minor compared to the advantages.
F.14 Improvements to existing production methods and tools or processes
COBISS.SI-ID: 2958193Gilbert's syndrome is the most common hereditary disorder of bilirubin metabolism. The causative mutation in Caucasians is almost exclusively a (TA) dinucleotide insertion in the UGT1A1 promoter. Affected individuals are homozygous for the variant promoter and have 7 TA repeats instead of 6. Promoters with 5 and 8 TA repeats also exist but are extremely rare in Caucasians. The aim of our study was to develop a high-performance liquid chromatography (DHPLC) assay for genotyping UGT1A1(TA)n polymorphism and to compare it with a previously described single-strand conformation polymorphism(SSCP) assay. Materials and methods: Fifty DNA samples with commongenotypes ((TA)6/6, (TA)6/7, (TA)7/7) as well as 7 samples with one of the following rare genotypes - (TA)5/6, (TA)5/7, (TA)6/8 or (TA)7/8 were amplified by polymerase chain reaction (PCR) and genotyped by DHPLC using sizing mode. All samples were previously genotyped by SSCP assay which was validated by sequencing analysis. Results: All samples with either common or rare genotypes showed completely concordant results between DHPLC and SSCP assays. Our results show that sizing DHPLC assay is more efficient compared to classical SSCP assay due to shorter time of genotyping analysis, ability of genotyping increased number of samples per day, higher robustness, reproducibility and cost-effectiveness with no loss of accuracy in detection of all UGT1A1(TA)n genotypes. Conclusions: We developed a new DHPLC assay which is suitable for accurate, automated, highthroughput, robust genotyping of all UGT1A1(TA)n polymorphism variants, compared to a labour intensive and time-consuming SSCP assay.
F.14 Improvements to existing production methods and tools or processes
COBISS.SI-ID: 3031153