Espinosa M I, Bertin A, Squeo F A, Cortés A, Gouin N
Departamento de Biología, Facultad de Ciencias, Universidad de La Serena, La Serena, Chile
Departamento de Biología, Facultad de Ciencias, Universidad de La Serena, La Serena, Chile.
Genet Mol Res. 2015 Jan 23;14(1):400-6. doi: 10.4238/2015.January.23.13.
Feces-based population genetic studies have become increasingly popular. However, polymerase chain reaction (PCR) amplification rates from fecal material vary depending on the species, populations, loci, and extraction protocols. Here, we assessed the PCR amplification success of three microsatellite markers and a segment of the mitochondrial control region of DNA extracted from field-collected feces of guanaco (Lama guanicoe) using two protocols - Qiagen DNA Stool Kit and 2 cetyltrimethylammonium bromide/phenol:chloroform:isoamyl alcohol (2CTAB/PCI) method. Chelex resin treatment to remove inhibitors was also tested. Our results show that the mitochondrial locus was the most difficult to amplify. PCR success rates improved for all markers after Chelex treatment of extracted DNA, and 2CTAB/PCI method (95.83%) appeared to perform slightly better than stool kit (91.67%) for the nuclear markers. Amplification success was significantly influenced by the extraction method, Chelex treatment, and locus (P < 0.001) but not by the freshness of the feces (fresh vs old, P = 0.17). The repeatability levels were high without Chelex treatment (> 0.89), but they decreased slightly after treatment for amplification of nuclear markers and markedly after treatment for amplification of the mitochondrial control region. Thus, we showed that Chelex treatment gives high PCR success, especially for nuclear markers, and adequate DNA extraction rates can be achieved from L. guanicoe feces even from non-fresh fecal material. Although not significant, 2CTAB/PCI method tended to provide higher successful amplification rates on a whole set of samples, suggesting that the method could be particularly useful when using small sample sizes.
基于粪便的群体遗传学研究越来越受欢迎。然而，粪便样本的聚合酶链反应（PCR）扩增率因物种、种群、基因座和提取方案而异。在这里，我们使用两种方案——Qiagen DNA粪便试剂盒和2%十六烷基三甲基溴化铵/苯酚：氯仿：异戊醇（2CTAB/PCI）法，评估了从野外采集的原驼（Lama guanicoe）粪便中提取的DNA的三个微卫星标记和线粒体控制区片段的PCR扩增成功率。还测试了用Chelex树脂处理以去除抑制剂的效果。我们的结果表明，线粒体基因座最难扩增。对提取的DNA进行Chelex处理后，所有标记的PCR成功率均有所提高，对于核标记，2CTAB/PCI法（95.83%）的表现似乎略优于粪便试剂盒（91.67%）。扩增成功率受提取方法、Chelex处理和基因座的显著影响（P < 0.001），但不受粪便新鲜度（新鲜与陈旧，P = 0.17）的影响。未经Chelex处理时重复性水平较高（> 0.89），但处理后核标记扩增的重复性略有下降，线粒体控制区扩增的重复性则显著下降。因此，我们表明Chelex处理可实现较高的PCR成功率，尤其是对于核标记，即使是从非新鲜粪便材料中也能从原驼粪便中获得足够的DNA提取率。虽然不显著，但2CTAB/PCI法在整套样本上往往能提供更高的成功扩增率，这表明该方法在使用小样本量时可能特别有用。
