Gagneux P, Boesch C, Woodruff D S
Department of Biology, University of California, San Diego, La Jolla 92093-0116, USA.
Mol Ecol. 1997 Sep;6(9):861-8. doi: 10.1111/j.1365-294x.1997.tb00140.x.
In the context of a study of wild chimpanzees, Pan troglodytes verus, we found that genotypes based on single PCR amplifications of microsatellite loci from single shed hair have a high error rate. We quantified error rates using the comparable results of 791 single shed hair PCR amplifications of 11 microsatellite loci of 18 known individuals. The most frequent error was the amplification of only one of the two alleles present at a heterozygous locus. This phenomenon, called allelic dropout, produced false homozygotes in 31% of single-hair amplifications. There was no difference in the probability of preferential amplification between longer and shorter alleles. The probability of scoring false homozygotes can be reduced to below 0.05 by three separate amplifications from single hairs of the same individual or by pooling hair samples from the same individual. In this study an additional 5.6% of the amplifications gave wrong genotypes because of contamination, labelling and loading errors, and possibly amplification artefacts. In contrast, amplifications from plucked hair taken from four dead individuals gave consistent results (error rate < 0.01%, n = 120). Allelic dropout becomes a problem when the DNA concentration falls below 0.05 ng/10 microL in the template as it can with shed hair, and extracts from faeces and masticated plant matter.
在一项对野生黑猩猩（指名亚种）的研究中，我们发现，基于从单根脱落毛发中对微卫星位点进行单重PCR扩增得到的基因型具有较高的错误率。我们利用18个已知个体的11个微卫星位点的791次单根脱落毛发PCR扩增的可比结果对错误率进行了量化。最常见的错误是在杂合位点仅扩增出两个等位基因中的一个。这种现象称为等位基因缺失，在31%的单根毛发扩增中产生了假纯合子。较长和较短等位基因之间的优先扩增概率没有差异。通过对同一个体的单根毛发进行三次单独扩增，或通过合并同一个体的毛发样本，将假纯合子的评分概率降低到0.05以下。在本研究中，另外5.6%的扩增由于污染、标记和加样错误以及可能的扩增假象而产生了错误的基因型。相比之下，从四只死亡个体拔取的毛发进行的扩增得到了一致的结果（错误率<0.01%，n = 120）。当模板中的DNA浓度低于0.05 ng/10 μL时，等位基因缺失就会成为一个问题，脱落毛发、粪便提取物和咀嚼过的植物物质都可能出现这种情况。
