| 摘要 |
FIELD: biotechnology.SUBSTANCE: invention relates to biotechnology and a set of synthetic oligonucleotide primers and method for use thereof. Proposed set comprises two pairs of primers having following structure - FIV F: 5′-AAGAGTCCCAAATATGCCATAGG-3′ and FIV R: 5′-TCCATCCAAATTGCTACTGTTC-3′; FeLV F: 5′-GAATAAACCTCTTGCTGTTTGC-3′ and FeLV R: 5′-AATCAGATCGAATGACAGAGACAC-3′. Presented primers do not contain palindromic repetitions of nucleotides, do not form expressed secondary structure and do not have extended G-C portions, and temperature of annealing is 55 °C for both pairs of oligonucleotides. Described method involves preparation of reaction mixture by mixing buffer of 10-fold for PCR - 2.5 mcl, a dNTP (25 mM) - 0.4 mcl, primers FIV F, FIV R, FeLV F and FeLV R (15 pmol/mcl) in 1 mcl, Taq polymerase (5 units/mcl) - 0.2 mcl MgCl(50 mM) - 1.5 mcl bidistilled water - 11.4 mcl and test DNA 5 mcl, amplification is carried out in following mode: total denaturation 95 °C - 3 minutes, cycle denaturation (95°C - 20 or 60 s) - annealing (55 °C - 20 or 60 s) - elongation (72 °C - 40 or 60 s), cycle of denaturation-annealing-elongation is repeated 35 times, final elongation 72 °C - 5 minutes, storing 4 °C - 10 minutes, and detection of obtained results is carried out by 1.5 % agarose gel. Presence or absence of amplified fragments of nucleotide sequences length 397 base pairs for FIV (feline immunodeficiency virus) and 221 base pairs for FeLV (feline leukemia virus) indicates presence or absence of viruses in organism cat.EFFECT: invention can be used in scientific research for detecting a genetic material of immunodeficiency viruses - FIV and leukemia FeLV cats by multiplex polymerase chain reaction (PCR) in animal blood.2 cl, 3 dwg, 3 tbl |
