Identification of genetic markers of predisposition to thrombogenic diseases by minisequencing analysis: reagent set “SNP2-TMG”

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Abstract

Thrombogenic risk factors (blood coagulation disorders and thrombophilia) are the cause of cardiovascular diseases, among which genetic factors are worth highlighting (genetic polymorphism of the blood coagulation system, angiogenesis factors, components of the lipid metabolism system). Early identification of clinically significant polymorphisms in genes that cause predisposition to thrombogenic diseases allows for preventive measures and timely diagnosis even before the onset of the clinical picture of the disease, and for patients with an already confirmed diagnosis genetic diagnostics makes it possible to check the hereditary nature of the disease, select treatment tactics, predict the risk of developing of adverse drug reactions. This article describes the process of developing the “SNP2-TMG” kit, designed to identify ten genetic markers of susceptibility to thrombogenic diseases (rs1801131, rs6025, rs11549465, rs429358, rs7412, rs1799963, rs6050, rs1799762, rs2010963, rs1801133), by minisequencing technique (SNaPshot technology). This kit has passed clinical trials and is approved for medical use.

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About the authors

А. V. Grudo

State scientific institution “Institute of Bioorganic Chemistry of the National Academy of Sciences of Belarus”

Author for correspondence.
Email: vasilevskaya.av@gmail.com
Belarus, Minsk

I. V. Haidukevich

State scientific institution “Institute of Bioorganic Chemistry of the National Academy of Sciences of Belarus”

Email: vasilevskaya.av@gmail.com
Belarus, Minsk

G. V. Sergeev

State scientific institution “Institute of Bioorganic Chemistry of the National Academy of Sciences of Belarus”

Email: vasilevskaya.av@gmail.com
Belarus, Minsk

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3. Fig. 1. Electrophoretic separation of amplification products of individual markers and multiplex reaction of Multiplex 1 of the SNP2-TMG kit in 10% polyacrylamide gel. “–” – negative control; 1 – rs7412 + rs429358; 2 – rs11549465; 3 – rs6025; 4 – rs1801131; 5 – multiplex reaction 1; St. – DNA fragment length standard 100–1000 bp (No. SM0241, Thermo Scientific).

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4. Fig. 2. Electrophoretic separation of amplification products of individual markers and multiplex reaction of Multiplex 2 of the SNP2-TMG kit in 10% polyacrylamide gel. “–” – negative control; 1 – rs2010963; 2 – rs6050; 3 – rs1799963; 4 – rs1801133; 5 – rs1799762; 6 – multiplex reaction 2. St. – DNA fragment length standard of 100–1000 bp (No. SM0241, Thermo Scientific).

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5. Fig. 3. Multiplex PCR 1 and 2 with different initial concentrations (1–20 ng) of human DNA in the reaction mixture. “–” – negative control, St. – DNA fragment length standard of 100–1000 bp (No. SM0241, Thermo Scientific).

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6. Fig. 4. Result of multiplex minisequencing of an arbitrary DNA sample using Plex SSHOT1 (a) and Plex SSHOT2 (b). Colored bars indicate bins of each possible nucleotide.

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