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A Limansky1 2 , O Limanskaya1
Institute of Clinical and Experimental Veterinary Medicine, Laboratory of Leukosis, Kharkov, Ukraine 1
I. Mechnikov Scientific Research Institute of Microbiology, Laboratory of Experimental and Applied Diagnostics, Kharkov, Ukraine 2
There are several methods for sexing embryos including detection of H-Y antigen, (male-specific transplantation antigen named histocompatibility-Y or H-Y), cytogenetic analysis, measurement of X-linked enzymes before Barr body formation, Y-chromosome specific probes, polymerase chain reaction (PCR). PCR amplification of sex-specific DNA sequences has been used to determine mammalian embryonic sex. The main goal of our work is creation of method for PCR test-system construction. We have developed this method for PCR sexing cattle embryos. A dominant factor provided by Y-chromosome has been required for mammals sex determination. The SRY-gene corresponds to this key factor and it has highly conserved motif for mammalian species. Computer analysis of Y-chromosome SRY-gene isolates conservative fragments is need to create test-system for SRY-gene PCR detection. We made multiple alignment and its statistic analysis for different mammals SRY isolates from GenBank: Bos taurus, Ovis aries, Sus scrofa, Capra hircus. The key parameter deter-mining PCR specificity is primer homology degree. Therefore SRY fragments with 100 % homology degree have been chosen for subsequent analysis and determination of primers system.
We've designed two sets of consensus primers for different PCR variants including nested PCR. These male specific primers have 100 % homology degree for cattle, sheep, goat SRY and one mismatch for pig SRY. Blood and cells lysates from 60-120 cells cattle embryos were used for amplification. The specificity of male and fe-male amplifications was 100 %. Nonspesific reactions were minimized by optimizing of PCR parameters. PCR amplification of SRY-fragments resulted in two bands on agarose gel. Internal primers set SRY2-SRY3 allows to amplify fragment of 176 bp long, external SRY1-SRY4 – 416. Sex determination was successful for all tested blood samples of cattle. For cattle blastomeres electrophoresis showed band of 176 bp for 3 samples from 10.
In conclusion we can state that designed test-system for mammalian embryos sexing has 100 % specificity.
This abstract is being presented on Monday, August 2 at 8:00 AM to 10:15 AM at CUB 2nd Floor Ballroom.