Allele-specific PCR with fluorescently labeled probes: criteria for selecting primers for genotyping.

Single-nucleotide polymorphisms (SNPs) can serve as reliable markers in genetic engineering, selection, screening examinations, and other fields of science, medicine, and manufacturing. Whole-genome sequencing and genotyping by sequencing can detect SNPs with high specificity and identify novel variants. Nonetheless, in situations where the interest of researchers is individual specific loci, these methods become redundant, and their cost, the proportion of false positive and false negative results, and labor costs for sample preparation and analysis do not justify their use. Accordingly, accurate and rapid methods for genotyping individual alleles are still in demand, especially for verification of candidate polymorphisms in analyses of association with a given phenotype. One of these techniques is genotyping using TaqMan allele-specific probes (TaqMan dual labeled probes). The method consists of real-time PCR with a pair of primers and two oligonucleotide probes that are complementary to a sequence near a given locus in such a way that one probe is complementary to the wild-type allele, and the other to a mutant one. Advantages of this approach are its specificity, sensitivity, low cost, and quick results. It makes it possible to distinguish alleles in a genome with high accuracy without additional manipulations with DNA samples or PCR products; hence the popularity of this method in genetic association studies in molecular genetics and medicine. Due to advancements in technologies for the synthesis of oligonucleotides and improvements in techniques for designing primers and probes, we can expect expansion of the possibilities of this approach in terms of the diagnosis of hereditary diseases. In this article, we discuss in detail basic principles of the method, the processes that influence the result of genotyping, criteria for selecting optimal primers and probes, and the use of locked nucleic acid modifications in oligonucleotides as well as provide a protocol for the selection of primers and probes and for PCR by means of rs11121704 as an example. We hope that the presented protocol will allow research groups to independently design their own effective assays for testing for polymorphisms of interest.

[Leukocyte telomere length and response to antiangiogenic therapy in patients with neovascular age-related macular degeneration.]

Age-related macular degeneration (AMD) is becoming the leading cause of vision loss in people over 60 years of age. The neovascular form of AMD (nVMD) is characterized by choroidal neovascularization (CNV), the main trigger of which is vascular endothelial growth factor (VEGF), the inhibition of which is the current standard of treatment. Significant variability of response to anti-VEGF therapy determines the relevance of the search for biological markers - prognostic criteria of treatment response. We analyzed the response of 110 nVMD patients to anti-VEGF therapy depending on the functional and anatomical parameters of the retina (according to optical coherence tomography, OCT) and leukocyte telomere length (LTL, was assessed by quantitative PCR). Positive dynamics of best corrected visual acuity (BCVA) was observed in 100% of eyes. The central retinal thickness (CRT) decreased after the 3rd injection to 265 [234-306] µm, by the end of the observation period - to 211 [190-262] µm. The retention of activity of the subretinal neovascular membrane (SNM) at the end of the observation period correlated with lower values of the initial BCVA and high values of the initial CRT. An association of LTL with response to treatment was revealed: in patients with higher LTL the active form of SNM was more often switched to inactive after three injections, while with lower LTL, the activity of SNM was more often preserved, which determined the need for more intravitreal injections.

[Assessment of mitochondria-associated genes for single nucleotide polymorphisms linked to the development of hypertrophic cardiomyopathy in the senescence-accelerated OXYS rats.]

Hypertrophic cardiomyopathy (HС) is a heterogeneous myocardial disease with a wide range of clinical manifestations and risk of development increasing with age along with myocardial changes characteristic of aging. The contribution of genetic component to the development of HC is obvious, however, the etiology and pathogenesis of this disease remains unclear in 50% of cases. The aim of the present study was to search for single nucleotide polymorphisms (SNPs) in mitochondria-associated genes that can contribute to the development of myocardial hypertrophy using RNA-Seq data from senescence-accelerated OXYS rats. Here we revealed SNPs with a possible negative effect on the function of the protein product in mitochondria-associated genes Fbxl4 and Slc25a32, mutations in which were not previously associated with HC. Alterations in the expression of these genes in the myocardium of OXYS rats at different stages of the development of pathological changes indicate that the revealed SNPs can contribute to the development of HC. Thus, SNPs in the Fbxl4 and Slc25a32 genes, as well as the genes themselves, can be considered promising molecular targets in the studies of the contribution of mitochondrial dysfunction to the development of myocardial hypertrophy.