绵羊早期胚胎性别鉴定巢式PCR体系的建立及其灵敏度研究

Sexing of early sheep embryos by using nested polymerase chain reaction

根据绵羊SRY(sex determining region of the Y)基因的723 bp的高度保守序列设计跨度为193 bp的两对巢式雄性特异引物,同时根据绵羊β-B-珠蛋白基因设计跨度为278 bp的两对巢式引物为内标引物,通过析因设计法建立了巢式PCR体系,对公、母羊肝组织基因组进行巢式PCR扩增,经1.5%的琼脂糖电泳检测,公羊同时出现278 bp的β-B-珠蛋白基因扩增带和193 bp的雄性特异扩增带两条带,而母羊只扩增出278 bp常染色体基因序列扩增带.用此巢式PCR体系扩增淋巴细胞,灵敏度≥10 pg DNA(约3个细胞),鉴定全程不超过130 min.

Two pairs of nested primers with the length of 193 bp were designed to identify the sex of early embryos based on the male specific SRY genes. Meanwhile, a pair of 278 bp primers were designed based on the sheep beta-B-globin genes on the autosome. The amplified product of the nested PCR by the 1.5 % agarose mini-gel electrophoresis showed that the male samples not only amplified a 193 base pair band but also 278 base pair band; the female samples amplified only a 278 base pair band. The developed nested PCR method was applied successfully with the sensitivity≥10 pg DNA （about 3 lymphocytes）,and the identification processes was no longer than 130 minites.