Influence of reagents used in bovine embryo biopsy on successful PCR sexing

The effects of bovine serum albumin (BSA), calf serum (CS), polyvinylpyrrolidone (PVP) and sucrose (Suc), commonly used in embryo biopsy, were first assessed with bovine purified DNA as templates under the standard PCR conditions using a kit for sexing of bovine embryos, When PCR amplification was performed in the reaction mixtures with different concentrations of BSA, PVP and Suc, a successful sex determination of purified DNA of each sex was made, regardless of any concentrations that we examined. These results indicate that the amount of these regents that we have used in actual embryo biopsy has no detrimental effects on PCR amplification. In contrast, when 20 pg of purified male DNA was subjected to PCR, more than 0.05% of CS in reaction mixture yielded the faint male band and 2.0% of CS interfered with the amplification process. However, the sex of 200 pg of purified DNA of each sex was successfully determined at concentration of 0.05-2.0% of CS. In a series of experiments in which the PB1 concentration in the reaction mixture was increased from x 0.1 to x 0.4, addition of ultra-pure water into the reaction mixture was varied in order to standardize the total reaction volume. When PCR amplification of 20 pg of purified male DNA was performed in the reaction mixture contained x 0.3 PB1, amplification itself was inhibited as evidenced by the lack of both male-specific and bovine-specific bands. Even when 200 pg of purified male DNA was subjected to PCR, x 0.3 PB1 in reaction mixture was not detected male-specific band. In this study, at least two factors are considered important in achieving a successful sex determination by PCR: one is avoidance of the use of calf serum when possible and another is proper concentrations of the various compounds present in the reaction mixture in which the amplification is performed