牛早期胚胎性别鉴定PCR反应体系的优化研究

The Study on Optimizing the System of PCR for Sex Determination of Bovine Preimplantation Embryos

根据牛SRY基因序列设计合成2对巢式PCR引物作为性别鉴定引物,根据牛酪蛋白基因序列设计了一对引物作为内标引物建立了牛胚胎性别鉴定的PCR反应体系。同时对常规PCR和巢式PCR在牛早期胚胎性别鉴定中的实用性进行比较。15头公牛、13头母牛的DNA样品检测结果表明:使用巢式PCR公牛可以扩增出205bp的SRY基因片段和403bp的酪蛋白基因片段,母牛只能扩增出403bp的酪蛋白基因片段;而使用常规PCR时公牛扩增出255bp的SRY基因片段和403bp的酪蛋白基因片段,母牛只能扩增出403bp的酪蛋白基因片段,其性别鉴定结果和实际完全一致。由于巢式PCR只需10个细胞就可以在紫外透射分析仪下看到扩增结果,而常规PCR则需要20～30个细胞,所以胚胎性别鉴定时使用巢式PCR效果更好。试验采用巢式PCR鉴定了10个奶牛胚胎的性别,同时还对血清是否会对试验结果产生影响进行了研究。

We designed 2 pairs of SRY gene nested primers for sex determination and a pair of casein protein gene primer as an internal standard in this study and established the system of PCR .At the same time,we compared the efficiency of nested PCR in sex determination of bovine embryos with that of normal PCR.DNA from 15 male and 13 female embryos were used as template in the PCR.As a result,205bp and 403bp bands could be visible on the gel in ultraviolet transilluminator by nested PCR for male,but for female,only 403bp band could be visible.By normal PCR,if DNA was from the male embryos,it could be amplified 255bp and 403bp products.From the female,it had only 403bp product.In every case the results were as expected for both female and male.It needed only 10 cells for obtaining amplification result by nested PCR,but 20 to 30 cells were needed by normal PCR.So we could obtain better results of sex determination of bovine preimplantation embryos by nested PCR.In this experiment,the sex of 10 bovine embryos was determined by nested PCR.We also researched whether the fetal calf serum could contaminate the embryo samples or not.