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美洲小鹿(偶蹄目,鹿科)粪便样本中线粒体、微卫星和牙釉蛋白基因座的可扩增性

Amplifiability of mitochondrial, microsatellite and amelogenin DNA loci from fecal samples of red brocket deer Mazama americana (Cetartiodactyla, Cervidae).

作者信息

Oliveira M L, Duarte J M B

机构信息

Núcleo de Pesquisa e Conservação de Cervídeos, Universidade Estadual Paulista "Júlio de Mesquita Filho", Jaboticabal, SP, Brasil.

出版信息

Genet Mol Res. 2013 Jan 16;12(1):44-52. doi: 10.4238/2013.January.16.8.

Abstract

We tried to amplify mitochondrial, microsatellite and amelogenin loci in DNA from fecal samples of a wild Mazama americana population. Fifty-two deer fecal samples were collected from a 600-ha seasonal semideciduous forest fragment in a subtropical region of Brazil (21°20'S, 47°17'W), with the help of a detection dog; then, stored in ethanol and georeferenced. Among these samples 16 were classified as "fresh" and 36 as "non-fresh". DNA was extracted using the QIAamp(®) DNA Stool Mini Kit. Mitochondrial loci were amplified in 49 of the 52 samples. Five microsatellite loci were amplified by PCR; success in amplification varied according to locus size and sample age. Successful amplifications were achieved in 10/16 of the fresh and in 13/36 of the non-fresh samples; a negative correlation (R = -0.82) was found between successful amplification and locus size. Amplification of the amelogenin locus was successful in 22 of the 52 samples. The difficulty of amplifying nuclear loci in DNA samples extracted from feces collected in the field was evident. Some methodological improvements, including collecting fresh samples, selecting primers for shorter loci and quantifying the extracted DNA by real-time PCR, are suggested to increase amplification success in future studies.

摘要

我们试图对来自野生美洲马萨马种群粪便样本中的线粒体、微卫星和牙釉蛋白基因座进行扩增。在一只探测犬的协助下,从巴西亚热带地区(南纬21°20′,西经47°17′)一片600公顷的季节性半落叶林片段中收集了52份鹿粪便样本;然后,将样本保存在乙醇中并进行地理定位。在这些样本中,16份被归类为“新鲜”样本,36份为“非新鲜”样本。使用QIAamp(®) DNA粪便迷你试剂盒提取DNA。52份样本中的49份扩增出了线粒体基因座。通过PCR扩增了5个微卫星基因座;扩增成功率因基因座大小和样本年龄而异。新鲜样本中有10/16成功扩增,非新鲜样本中有13/36成功扩增;成功扩增与基因座大小之间存在负相关(R = -0.82)。52份样本中有22份成功扩增出牙釉蛋白基因座。从野外采集的粪便中提取的DNA样本扩增核基因座存在困难是显而易见的。建议采取一些方法改进措施,包括采集新鲜样本、选择较短基因座的引物以及通过实时PCR对提取的DNA进行定量,以提高未来研究中的扩增成功率。

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