Fredsted T, Villessen P
Department of Ecology and Genetics, Institute of Biological Sciences, University of Aarhus, Aarhus, Denmark.
Am J Primatol. 2004 Nov;64(3):345-50. doi: 10.1002/ajp.20083.
Molecular sexing of mammals is normally done by PCR amplification of Y chromosomal fragments, or coamplification of homologous fragments from both sex chromosomes. Existing primers are often unreliable for distantly related species due to mutations in primer regions. Currently there are no published primers for the sexing of prosimian DNA. We show that an existing method (using the zinc finger protein) based on a size difference between the X and Y homologs does not work in prosimians. Multiple alignments of distantly related mammalian species from Genbank and genome databases enabled us to identify conserved regions in the amelogenin gene. Using these conserved regions, we can target species that have no sequence information. We designed a single, conserved primer pair that is useful for fast and reliable molecular sexing of prosimian primates. A single PCR yields two fragments in males and only one in females, which are easily separated with the use of agarose gels. Amplification of separable fragments was successful in seven species of lemurs, as well as humans.
哺乳动物的分子性别鉴定通常通过对Y染色体片段进行PCR扩增，或对来自两条性染色体的同源片段进行共扩增来完成。由于引物区域的突变，现有的引物对亲缘关系较远的物种往往不可靠。目前尚无已发表的用于原猴亚目DNA性别鉴定的引物。我们发现，基于X和Y同源物之间大小差异的现有方法（使用锌指蛋白）在原猴亚目中不起作用。通过对来自Genbank和基因组数据库的亲缘关系较远的哺乳动物物种进行多重比对，我们能够鉴定牙釉蛋白基因中的保守区域。利用这些保守区域，我们可以针对没有序列信息的物种。我们设计了一对单一的保守引物，可用于快速、可靠地对原猴亚目灵长类动物进行分子性别鉴定。单次PCR在雄性中产生两个片段，在雌性中仅产生一个片段，通过使用琼脂糖凝胶很容易将它们分开。在七种狐猴以及人类中，均可成功扩增出可分离的片段。
