SULM – Schweizerische Union für Labormedizin | Union Suisse de Médecine de Laboratoire | Swiss Union of Laboratory Medicine

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M. Beranek1 , J. Horacek2 , V. Palicka1

1Institute of Clinical Biochemistry and Diagnostics, Charles University Hospital, Sokolska 581, 50005 Hradec Kralove, Czech Republic; Cette adresse e-mail est protégée contre les robots spammeurs. Vous devez activer le JavaScript pour la visualiser., 2Prague Psychiatric Center, 3rd Faculty of Medicine of Charles University, Center of Neuropsychiatric Studies, Prague, Czech Republic

Catechol-O-methyltransferase (COMT; EC 2.1.1.6) is a biotransformation enzyme of catechols that plays an important role in the metabolism of noradrenaline, adrenaline, and dopamine. The COMT gene maps to chromosome 22q11.2. A transition G to A at a polymorphic codon 108/158 causes a valine to methionine substitution resulting in a thermolabile form of COMT with 3-4-fold reduction of catalytic activity altering the metabolism of catecholamine neurotransmitters. Here we present a rapid real-time PCR method for COMT genotyping via melting point analysis with fluorescent hybridization probes. Each reaction (10 ul)contained 5 pmol of primers, 1 pmol each of hybridization probes, 2 mmol/l MgCl2, 1 ul of LightCycler FastStart DNA Master Hybridization Probes (Roche), and 50–100 ng of genomic DNA. The probes were designed using Roche LightCycler Probe Design Software. After 45 cycles of amplification (95 C for 2 s, 55 C for 5 s, and 72 C for 5 s), melting analysis was performed by denaturation at 95 C for 30 s, annealing at 40 C for 90 s, and slow heating to 75 C with a ramp rate of 0.1 C/s. The homozygous Met/Met genotype showed a melting temperature peak of 59 C, the homozygous Val/Val type presented a melting peak at 64 C, and the heterozygous genotype Met/Val showed two peaks at 59 and 64 C. The genotyping was performed on DNA samples extracted from peripheral blood cells of 118 Czech Caucasians. The reliability of the results was confirmed by a PCR/RFLP method described previously. Both techniques gave identical results. 49% of subjects were heterozygous Met/Val, 21% were homozygous Met/Met, and 30% were homozygous Val/Val. The estimation of Val-108/158 allele frequency was about 54%. Detection of a single-nucleotide polymorphism at COMT codon 108/158 by real-time PCR could be an attractive approach providing rapid and accurate results of DNA analysis, useful namely for larger population studies.

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