M.N. Karagyaur1, K.D. Bozov2, A.L. Primak3, S.S. Dzhauari4, M.E. Illarionova5, P.S. Klimovich6, E.V. Semina7, L.M. Samokhodskaya8, V.S. Popov9, D.A. Sheleg10, E.A. Neyfeld11

1–11 FSBEI HE “Lomonosov Moscow State University” (Moscow, Russia)

1 m.karagyaur@mail.ru, 2 kir-bozov@yandex.ru, 3 primak.msu@mail.ru, 4 stalik.djauari@yandex.ru, 5 mar729i63illar90@yandex.ru, 6 lex2050@mail.ru, 7 e-tal@yandex.ru, 8 samokhodskay@gmail.com, 9 galiantus@gmail.com, 10 sheleg-da@mail.ru, 11 ea.neyfeld@mail.ru

Identification of genomic variants and elucidation of their functional significance is one of the tasks of human genetics. Allele-specific PCR technology, especially ARMS modification, is best suited for solving this task in large-scale routine studies. The problem is the lack of understandable and accessible algorithms for the design of 3'-end primers that determine the specificity and efficiency of ARMS analysis.

Purpose of work – to develop an algorithm for the design of 3‘-ends of ARMS primers for the detection of single nucleotide genomic variants; develop a software package to automate the design of 3’-end ARMS primers.

An algorithm for the design of 3'-ends of internal ARMS primers was developed, and in turn it was used to create the ARMS PrimerDesign software. The developed software allows optimizing the design of ARMS primers regardless of the nature of the nucleotide substitution, as well as the orientation of primers relative to the target genomic variant. Using the ARMS PrimerDesign software we designed primers for detection of genomic variants #rs4760 (PLAUR gene), #rs17445840 (CDH2 gene), #rs28561984 (DCHS2 gene), #rs10999947 (CDH23 gene), previously identified in DNA samples of patients with mental disorders. The specificity of ARMS-analysis with primers obtained was assessed by comparing the ARMS results with the results of Sanger sequencing. We found that nucleotide «-2» substitution in internal primers increases the efficiency of ARMS-analysis compared to nucleotide «-1» substitution, but decreases its specificity in some cases: some homozygous variants may be misinterpreted as heterozygous. It has been found experimentally that the optimal annealing temperature for ARMS PCR can be 5-7⁰C higher than theoretically predicted.

The results obtained confirm the high specificity and efficiency of using ARMS, a kind of allele-specific PCR, for routine detection of desired genomic variants in large sample collections. The optimal specificity and efficiency of ARMS can be achieved by making nucleotide substitutions in the «-1» position at the 3'-end of internal allele-specific primers, which can be attained with the help of the ARMS PrimerDesign software developed by us, followed by experimental determination of the optimal annealing temperature for the tetraplex of designed primers.

Karagyaur M.N., Bozov K.D., Primak A.L., Dzhauari S.S., Illarionova M.E., Klimovich P.S., Semina E.V., Samokhodskaya L.M., Popov V.S., Sheleg D.A., Neyfeld E.A. Optimization of allele-specific PCR technology for detection of single nucleotide genomic variants. Technologies of Living Systems. 2025. V. 22. № 2. Р. 37-48. DOI: https://doi.org/10.18127/j20700997-202502-04 (In Russian).

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