The Study On Sex Determination Of Bovine Embryos By Polymerase Chain Reation (PCR)

In modern cattle breeding,people want to according their desire to control the sex ratio of cattle,rapid breeding speed and expand the use of good cow,so the determination of bovine embryos sex has become an important subject. Now , on the base of traditional cytogenetic analysis ,in order to satisfied the practice need, scientists use modern technology improve the accurate of sexing and shorten the time-In this experiment,we successfully isolated genome DNA from bovine peripheral blood.With the application of PCR, we amplified bovine Y chromosome specific DNA fragment and optimum the PCR condition of sexing. Then using PCR we amplified ,cloned,sequencedand analyzed bovine SRY gene. The test obtained the results and advances as follows:1. we have established the method that could isolate good quality genome DNA from peripheral blood.2. The study has established a rapid and reliable consecutive and multiplex PCR method for the sexing of bovine early embryos.That is,two pairs of DNA sequences were selected for amplification of male- and bovine-specific DNA.respectively.The method attained that two gene fragments amplifiction in one PCR tube.We have optimumed PCR conditions,?consecutive and mutiplex PCR,the first cycles were done with male-specific primers followed by an additional cycles with bovine-specific primers to attain the unequal number of copies of the two repetitive sequences.The method not noly assured strong specific and high sensitive,but also make the sexing course completed within 2 hours.The sexing efficiency was 95.24%.we have set up an effective PCR method sexing bovine embryos in farm condition.3. We have utilized PCR method amplified ,cloned,sequencedand analyzed bovine SRY gene . With the existence of a pair of specific primers that bovine genome DNA was used as template in PCR, we have optimum the PCR conditiones.The PCR product was purified using a UNIQ-10 Column DNA Gel Extraction Kit, then was cloned into a pMD18-T vector.After being transformed into E.coli.,isolated plasmids DNA,and tested by restriction endonuclease digestion ,the positive clone was sequenced.The result indicated that the size of the fragment cloned is 2713bp,and its sequence homology with the designed one is 98.7%and 98.1% respectively. BLAST result showed that there are some levels homology with other animals , which implicated that SRYgene keep conservation in evolution courses. The result not only and offered materials for the study of sex determation,but also established the basis for the deep study of order control.sex determination mechanism and evolution of SRY gene. The study provided the foundition for designing specific primers in order to identification animal sex,that could make sexing more accurate and specific.