OPTIMIZATION OF THE TETRA-PRIMER ARMS-PCR METHOD FOR DIAGNOSING SUBFERTILITY SYNDROME IN BULLS

Abstract

The authors of the work optimized the conditions for conducting the TETRA-PRIMER ARMS-PCR reaction for the detection of carriers of the subfertility syndrome in bulls of different breeds of foreign and domestic selection. Experimentally determined the optimal concentration of forward and reverse external and internal primers in the amount of 1,0 µl of each primer with a final volume of the reaction mixture of 25,0 µl. The following temperature regime was effective: initial denaturation at 95 °C – 5 min, at the first stage of amplification, the number of cycles was 17, denaturation at 94 °C – 30 sec, primer annealing at 68 °C – 30 sec, with a decrease in temperature by 1 °C for each cycle, elongation at 72°C – 30 sec, next 30 cycles denaturation at 94°C – 30 sec, primer annealing at 51°C – 30 sec and elongation at 72°C – 30 sec, final synthesis at 72°C -5 minutes. It has been established that a change in the concentration of magnesium chloride does not significantly affect the effectiveness of the TMEM95 gene amplification process. Thus, using the optimized TETRA-PRIMER ARMS-PCR method, only 223 breeding bulls from three breeding centers of the Republic of Kazakhstan were tested, one heterozygous carrier of the subfertility syndrome was identified, a sire of the Simmetal breed of foreign selection, the occurrence of the studied genetic defect in sires was low and amounted to 0.44%.