A SET OF OLIGONUCLEOTIDE PRIMERS AND PROBES FOR GENOTYPING POLYMORPHIC LOCUS OF DNA ASSOCIATED WITH THE RISK OF DEVELOPMENT OF SPORADIC FORM OF ALZHEIMER'S DISEASE IN RUSSIAN POPULATIONS

摘要

1. A set of oligonucleotide primers and probes for the analysis of genetic polymorphism at the ApoE (rs429358, rs7412), ApoJ (rs11136000), and GAB2 (rs231311000) DNA loci, which includes the following stages: multiplex "nested" two-stage PCR, in which a DNA sample is used as a template for amplification; when conducting PCR, a set of primers is used with the sequences shown in SEQ ID NO: 9-20; as a result, at the second PCR stage, a fluorescently labeled PCR product is formed; in the multiplex reaction of stage I, primers are used: 9, 10, 13, 14, 17, 18; in the multiplex reaction of stage II, primers are used: 11, 12, 15, 16 , 19, 20; the reaction mixture at the first PCR stage contains 5 pmol of each primer, 67 mM Tris_HCl, pH 8.6, 166 mM (NH4) 2SO4, 1.5 mM MgCl2, 0.2 mM each of dNTP, 2.5 units. Act. Taq polymerase and matrix in an amount of at least 10 ng of genomic DNA. The reaction is carried out in the following mode: preliminary DNA denaturation at 94 ° C (5 min), 35 amplification cycles according to the following scheme: 94 ° C, 30 s; 65 ° C, 30 s; 72 ° C, 30 s; further 72 ° C, 10 min. The second stage of PCR is carried out in a reaction mixture of the same composition, but instead of 5, 50 pmol of one of the primers is added with the addition of a fluorescent label to preferentially produce a fluorescently labeled single-chain PCR product. (B) immobilization of oligonucleotide probes, the sequences of which are presented in SEQ ID NO: 1-8; (c) - hybridization of the labeled PCR product with immobilized oligonucleotides presented in SEQ ID NO: 1-8; (d) - registration and interpretation of hybridization results. 2. A set of oligonucleotides for the identification of polymorphic fragments of genes amplified�