Exogenous contaminating DNA in Taq polymerases: A method to avoid false-positive results when detecting the blaTEM gene.

In the course of developing an assay to identify genes responsible for antibiotic resistance in gram-negative bacteria, it has been found that standard (not DNA-free) Taq DNA polymerases were contaminated with blaTEM gene fragments that varied in length and quantities. The complete blaTEM gene sequence was either absent or was detected in infinitesimal amounts. We developed an approach to avoid false-positive findings caused by contaminating blaTEM gene sequences in conventional polymerases. The method is based on selection of a target sequence to be detected within the blaTEM gene in such a way that the chosen sequence is amplified with primers incapable of amplifying contaminating DNA sequences of the polymerase.

Microarray with LNA-probes for genotyping of polymorphic variants of Gilbert's syndrome gene UGT1A1(TA)n.

BACKGROUND: Gilbert's syndrome is a common metabolic dysfunction characterized by elevated levels of unconjugated bilirubin in the bloodstream. This condition is usually caused by additional (TA) insertions in a promoter region of the uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1) gene, which instead of the sequence А(TА)6TАА contains А(TА)7TАА. While the condition itself is benign, it presents elevated risk for patients treated with irinotecan, a common chemotherapy drug. METHODS: The technique is based on hybridization analysis of a pre-amplified segment of the UGT1A1 gene promoter performed on a microarray. Specific probes containing locked nucleic acids (LNA) were designed and immobilized on the microarray to provide accurate identification. RESULTS: A microarray has been developed to identify both common and rare variants of UGT1A1(TA)n polymorphisms. In total, 108 individuals were genotyped. Out of these, 47 (43.5%) had homozygous wild-type genotypes (TA)6/(TA)6; 41(38%) were heterozygotes (TA)6/(TA)7; and 18 (16.7%)--homozygotes (TA)7/(TA)7. In two cases (1.8%), rare genotypes (TA)5/(TA)7 and (TA)5/(TA)6 were found. The results were in full agreement with the sequencing. In addition, synthetic fragments corresponding to all human allelic variants [(TA)5, (TA)6, (TA)7, (TA)8] were successfully tested. CONCLUSIONS: The developed microarray-based approach for identification of polymorphic variants of the UGT1A1 gene is a promising and reliable diagnostic tool that can be successfully implemented in clinical practice.