engleski

Allele-specific oligonucleotide PCR: detection of A/G dimorphism in intron 13 of human monoamine oxidase B (MAO-B) gene

Detection of a single base change can be, besides DNA sequencing, achieved by allele-specific oligonucleotide polymerase chain reaction (ASO PCR). The annealing of reverse primer with template depends, in our case, on complementarity of the last primer nucleotide with respective base in DNA sequence. In intron 13 of the gene encoding human monoamine oxidase B polymorphic site has been identified refered to as A/G dimorphism. The variant base (A or G), although located in a non coding region, is in proximity to the 3' splicing acceptor site of intron 13. Presumably this region could be involved in transcriptional regulation of the gene, thereby affecting the amount of expressed protein i. e. MAO-B activity. In our study, DNA extracted from peripheral blood leukocytes served as a template for allele-specific PCR. Regarding primers, the forward one was common for both reactions and the reverse primers were A- and G-specific as mentioned above. Hence, for each individual two separate PCRs were performed with A- or G-allele specific reverse primer (for A- or G-allele amplification). PCR products (663 bp long) were analysed by electrophoresis on 1.6% agarose gel containing ethidium bromide. Individuals homozygous for A- or G-allele have PCR products only in their respective PCRs whilst heterozygous individuals have products in both reactions. We are actually screening a large population of healthy individuals in order to get insight into frequency distribution of the MAO-B gene variants regarding the mentioned polymorphism. This should serve us as a reference for future molecular genetic studies on MAO-B gene in various neuropsychiatric disorders (e.g. migraine).

allele-specific PCR; monoamine oxidase B; gene; A/G dimorphism

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