Study On The Sex Identification System With Polymerase Chain Reaction In Sheep

The polymerase chain reactions (PCR) were established to identify the sex of sheep according to examine sex determination gene of sheep by using multiple PCR system,or by using nested polymerase chain reaction and the specificity of sex determining region of Y (SRY) primers, discussed the factors which affected PCR ampplication as well as their reciprocity. This paper's main content as follow:(1) Basing on the 723 base pairs sheep sex determining region of Y chromosome genes and the 3249 base pairs sheep beta-B-globin genes, two pairs of nested sex determination gene primers and two pairs of nested autosome gene primers were designed using primer-3.(2) Optimuming composition of these primers in order to establish the multiplex PCR and nest PCR system used to identify the sheep sex. With a 3×2×3 factorial experiment had determined the combination of concentrations of dNTP,Mg²⁺,and temperature. At the same time, Optimuming the dNTP, Mg²⁺, temperature, the procedure and parameter of nest PCR.(3) The gene of human, bovine, rabbit, lymphocytes buffer were amplified by the nest PCR system established in the paper in order to detect the specific bands and the possible pollution during the sex identification.The optimal multiple PCR system was: the concentration of Globin 559, SRY 337, Mg²⁺, dNTP, Taq DNA polymerase, 10×PCR Buffer was 0.4μM, 0.4μM, 2.0mM, 300μM, 1U, and 2.5μl, total volume of PCR was 25μl. Application procedure was: 94℃predegeneration 3min-{94℃degeneration 20s-58℃annealing 20s-72℃extension 20s} 30 cycles-72℃extension 3min-holding at 4℃. The optimal multiplex nested PCR system application accurate rate is 100% for male and female sheep genomic DNA, and the system was applied successfully with the sensitivity of 20 lymphocytes of male sheep.Nested PCR second application of two steps was done after 20 cycles of the optimal multiplex PCR amplification, and the optimal nested PCR second application system was: the concentration of Globin 278, SRY 193, Mg²⁺, dNTP, Taq DNA polymerase, 10×PCR Buffer was 0.4μM, 0.4μM, 2.0 mM, 200uM, 1U, and 2.5μl, total volume of PCR was 25ul.Application procedure was: 94℃predegeneration 3min-{94℃degeneration 20s-64℃annealing and extension 20s} 30 cycles-72℃extension 3min-holding at 4℃. The optimal multiplex and nested PCR system's sensitivity is 4 lymphocytes'DNA, and the identification processes was no more than 110 minutes.The primers of sheep SRY gene designed in this study are very specific beside cattle, the DNA of other animal (such as human, rabbit,etc) would not affect the sexing result.The experiment established nested PCR system's operation is simple, quick, and accurate rate is high, which can satisfy complately sex identification needing.