METHOD FOR SIMULTANEOUS GENODIAGNOSTIC OF FOUR MUTANT ALLELES OF KAPPA-CASEIN IN CATTLE AND TEST SYSTEM FOR IMPLEMENTATION THEREOF

摘要

FIELD: veterinary medicine.SUBSTANCE: group of inventions relates to molecular genetics and can be used in veterinary practice and animal husbandry for diagnosing four kappa-casein alleles. Disclosed is a method for simultaneous genodiagnostics of four mutant kappa-casein alleles in cattle, involving DNA recovery from biological material, polymerase chain reaction in real time, using three reaction mixtures containing 50 mM KCl, 50 mM TRIS-HCl, 250 nM dNTP, 2.5 mM MgCl, primers - in concentration of 200 nM, fluorescent-marked oligonucleotide samples - in concentration of 100 nM, diluent, Taq DNA polymerase, three positive and one negative control sample for each reaction mixture. In preparation for the reaction, the required volume of components is calculated based on the number of analyzed samples plus 4, the reagents are mixed, then in prepared for PCR test tubes are introduced by 20 mcl of prepared PCR-mixture, in each PCR test tube is added by 5 mcl of control and analyzed samples, test tubes are placed in a thermocycler, annealing of primers passes at cycling step at 64 °C for 30 s with the number of amplification cycles equal to 40, analysis of the obtained data is carried out by comparing the amplified gene sections, results are interpreted on the basis of presence or absence of intersection of the fluorescence curve with a threshold line installed at the corresponding level. Also disclosed is a test system for implementing said method.EFFECT: group of inventions enables diagnosing four alleles simultaneously; faster execution of PCR-PB; reduced labour intensity; possibility of monitoring accuracy of analysis.2 cl, 1 dwg, 4 tbl