Department of Agricultural Sciences, Biotechnology and Food Science, Cyprus University of Technology, 3603 Lemesos, Cyprus.
J Dairy Sci. 2010 Dec;93(12):5996-9. doi: 10.3168/jds.2010-3569.
In the present study, a rapid and cost-effective PCR-based assay was developed for the genetic identification of 2 different variants within intron 2 of the prolactin gene. This polymorphism has previously been associated with milk traits in some ovine breeds and was recently proposed as a potential marker for future breeding schemes in dairy sheep. Until now, 2 alleles (A and B) have been identified by PCR-RFLP that included HaeIII digestion of a 2.5-kb PCR fragment. By partial sequencing of the prolactin gene intron 2, it was found that the B variant results from a 23-bp deletion of the A variant of the prolactin gene and not from an extra HaeIII digestion site, as had been reported. This finding assisted the design of new primers for analysis of prolactin intron 2 variants based on the size of an easily amplified short PCR product, thereby avoiding the need and cost for additional digestions. The method was validated by genotyping 80 animals from 2 breeds and showed 100% sensitivity and specificity compared with the PCR-RFLP assay. The established simplified PCR assay was then successfully used to genotype 356 Chios sheep.
在本研究中，开发了一种快速且经济有效的基于 PCR 的检测方法，用于鉴定催乳素基因第 2 内含子中 2 种不同变体的遗传特征。该多态性先前与一些绵羊品种的牛奶性状有关，最近被提议作为未来奶绵羊育种计划的潜在标记。到目前为止，通过 PCR-RFLP 已经鉴定出 2 种等位基因（A 和 B），包括对 2.5kb PCR 片段进行 HaeIII 消化。通过对催乳素基因第 2 内含子的部分测序发现，B 变体是由于催乳素基因 A 变体的 23bp 缺失，而不是像之前报道的那样，是由于额外的 HaeIII 消化位点。这一发现有助于根据易于扩增的短 PCR 产物的大小设计用于分析催乳素内含子 2 变体的新引物，从而避免了额外消化的需要和成本。该方法通过对来自 2 个品种的 80 个动物进行基因分型进行了验证，与 PCR-RFLP 检测相比，其灵敏度和特异性均达到 100%。随后，建立的简化 PCR 检测方法成功地用于对 356 只奇奥绵羊进行基因分型。
