PCR扩增ZFY、ZFX序列鉴定奶牛性别的灵敏度研究

Sensibility of Sex Identification with Amplification of ZFY and ZFX Gene Sequences in Bovine by PCR

从克隆、测序所得奶牛ＺＦＹ、ＺＦＸ序列中设计１对高特异性引物（或探针），即分别特异于牛ＺＦＹ、ＺＦＸ的ＺＦ３、ＺＦ４，与克隆时使用的引物ＺＦ２搭配成ＺＦ２、ＺＦ３和ＺＦ２、ＺＦ４。利用这２对引物和ＺＦ１、ＺＦ２对奶牛基因组ＤＮＡ进行ＮｅｓｔＰＣＲ来判断性别，结果达到用超微量（８ｐｇ）ＤＮＡ，即可检出ＺＦＹ、ＺＦＸ序列而判定雌雄的灵敏度；用非同位素标记试剂盒（ＤＩＧ－Ｓｙｓｔｅｍ）标记ＺＦ３、ＺＦ４作为探针，对ＺＦ１、ＺＦ２扩增产物点杂交来判断性别。

The sequences of ZFY and ZFX gene fragments obtained from cloning and sequencing in bovine were analyzed. It was found that there were 88.7% homologous region within the two fragments. The remains appeared to be different. According to these different sequences, the primers or probe (ASO) ZF 3 specific for ZFY and the ZF 4 specific for ZFX were designed. One set of ZFY specific primers formed by ZF 2 and ZF 3 and the another set of ZFX specific primers formed by ZF 2 and ZF 4 were used to identify the sex of bovine by nest PCR. The results showed that a 8 pg DNA level of the sensibility for sex identification achieved in bovine by the nest PCR.