基于牙釉质基因巢式PCR性别鉴定 超微量DNA检测方法的建立

实验根据山羊牙釉质(AMEL)基因序列设计巢式引物,用紫外分光仪检测基因组DNA的浓度,梯度稀释(10 -2μg/μL、10-3 μg/μL,10-4μg/μL,10-5μg/μL),巢式PCR扩增后琼脂糖凝胶电泳检测.结果表明,利用巢式PCR方法对10 pg量基因组DNA(约3个细胞)进行扩增,得到明显目的条带.巢式...     

牙釉质基因鉴定山羊性别的研究

根据牙釉质(amelogenin,AMEL)同源基因在山羊X、Y染色体上存在不同程度碱基缺失的特点,设计1对引物,对山羊血液基因组DNA混合样进行PCR扩增,将PCR产物纯化、克隆并测序;然后对46个山羊血液DNA样本(♀:22个,♂:24个)进行性别鉴定。结果显示,雌性山羊样品经扩增只产生一条非特异性条带,雄性山羊样品产生一条非特异性条带和一条特异性条带;非特异性片段长度为349 bp,特异性片段为289 bp;46个山羊血液DNA样本鉴定结果与实际性别对比,雌性准确率为100%(22/22),雄性准确率为100%(24/24)。     

Sexing of pre-implantation ovine embryos through polymerase chain reaction-based amplification of GAPDH,SRY and AMEL genes

The ability to identify the sex of embryo and control of sex ratio has a great commercial importance to livestock industry. Prediction of embryonic sex could be useful in the management decisions of sex selection in breeding programs. Several methods have been attempted to determine the sex but the polymerase chain reaction (PCR)-based sexing method is generally favoured, as it is cost effective, simple and reliable. The aim of the present study was to identify sex of sheep embryos produced in vitro through amplification of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), sex-determining region Y (SRY) and amelogenin genes present in genomic DNA (gDNA) of embryos through PCR. To avoid false interpretation of the result by no amplification of SRY in female embryos, a duplex PCR was approached to amplify combinedly SRY and GAPDH genes. Sex-specific blood was used in PCR as positive control. In vitro sheep embryos were produced as per standardized protocol of laboratory. Sexing of sex-specific blood and in vitro produced embryos were approached though PCR to amplify the respective genes using gDNA present in the sample without its traditional isolation. The accuracy of sex prediction for embryos was 100% by this procedure.     

基于AMEL基因的绵羊性别鉴定技术

[目的]探讨将牙釉蛋白(AMEL)基因用于绵羊性别鉴定的可行性。[方法]采用酚-氯仿法从湖羊的耳皮肤组织中提取基因组DNA。分别以公羊和母羊的DNA为模板,以AMEL引物和SRY引物进行PCR扩增。[结果]经扩增后母羊得到一条来自X染色体的270bp条带,而公羊则得到270和210 bp的两条带。两种方法对10份已知DNA样品检测结果与实际性别完全一致。PCR产物的电泳条带较为清晰,相同性别电泳条带数完全一致,而不同性别间电泳条带数和片段大小差异显著。AMEL引物扩增雌性个体DNA实际电泳条带数和理论条带数一致,而来自雄性结果却不一致,这可能与非特异性扩增有关。[结论]AMEL基因比SRY基因具有更高的灵敏性,可用于判定XY染色体动物的性别。