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1. 发明授权

US5876942A Process for sexing cow embryos 失效
标题翻译：性交奶牛胚胎的过程
公开(公告)号：US5876942A
公开(公告)日：1999-03-02
申请号：US899811
申请日：1997-07-24
申请人： Winston Teng-Kuei Cheng , Chuan-Mu Chen , Che-Lin Hu , Chih-Hua Wang , Kong-Bung Choo
发明人： Winston Teng-Kuei Cheng , Chuan-Mu Chen , Che-Lin Hu , Chih-Hua Wang , Kong-Bung Choo
IPC分类号： C12Q1/68 , C07H21/02 , C07H21/04
CPC分类号： C12Q1/6879
摘要： A rapid, highly reproducible and sensitive technique has been successfully developed for sexing the cow embryos, by method of polymerase chain reaction (PCR) against the amelogenin (bAML) genes located on both X- and Y-chromosomes of the Holstein dairy cattle. Results from DNA sequence analysis showed that there was only 45% homology between the intron 5 of AMLX and AMLY genes. Based on these sequences a pair of sex-specific primers, pbAML5XY(+) and pbAML5XY (-),were designed allowing to amplify a single fragment of 476-bp from the female cattle and two fragments of 476-bp and 341-bp from the male ones, respectively. The most important feature is that the precise sensitivity of sex-determination was confirmed to be reached as minimum template as trace amount of genomic DNA content in either a single lymphocyte or a single blastomere isolated from cow embryo at day-6 to day-7. Moreover, neither those of complicated procedures for purifying the DNA prior the PCR nor any extra pair of primers for serving as internal control is thought to be essential and the sex-determination of over hundred embryos can be completed at once within 4hrs.
摘要翻译：通过聚合酶链反应（PCR）对位于荷斯坦奶牛的X射线和Y染色体上的牙釉蛋白（bAML）基因的方法，已经成功开发了快速，高度重现和敏感的技术，用于性别雌性胚胎。 DNA序列分析结果表明，AMLX的内含子5与AMLY基因之间只有45％的同源性。基于这些序列，设计了一对性别特异性引物pbAML5XY（+）和pbAML5XY（ - ），从而扩增了来自雌性牛的476-bp的单个片段和476bp和341bp的两个片段男性分别。最重要的特征是确定了在第6天至第7天从单纯淋巴细胞或从胚胎中分离的单个卵裂球中的最小模板作为最小模板作为痕量的基因组DNA含量。此外，在PCR前纯化DNA的复杂程序和用作内部对照的任何额外的引物对都不被认为是至关重要的，并且可以在4小时内立即完成100多个胚胎的性别测定。

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