Max Planck Institute for Evolutionary Anthropology, Deutscher Platz 6, D-04103 Leipzig, Germany.
Mol Ecol Resour. 2009 Jan;9(1):28-36. doi: 10.1111/j.1755-0998.2008.02387.x. Epub 2008 Oct 20.
Many studies in molecular ecology rely upon the genotyping of large numbers of low-quantity DNA extracts derived from noninvasive or museum specimens. To overcome low amplification success rates and avoid genotyping errors such as allelic dropout and false alleles, multiple polymerase chain reaction (PCR) replicates for each sample are typically used. Recently, two-step multiplex procedures have been introduced which drastically increase the success rate and efficiency of genotyping. However, controversy still exists concerning the amount of replication needed for suitable control of error. Here we describe the use of a two-step multiplex PCR procedure that allows rapid genotyping using at least 19 different microsatellite loci. We applied this approach to quantified amounts of noninvasive DNAs from western chimpanzee, western gorilla, mountain gorilla and black and white colobus faecal samples, as well as to DNA from ~100-year-old gorilla teeth from museums. Analysis of over 45 000 PCRs revealed average success rates of > 90% using faecal DNAs and 74% using museum specimen DNAs. Average allelic dropout rates were substantially reduced compared to those obtained using conventional singleplex PCR protocols, and reliable genotyping using low (< 25 pg) amounts of template DNA was possible. However, four to five replicates of apparently homozygous results are needed to avoid allelic dropout when using the lowest concentration DNAs (< 50 pg/reaction), suggesting that use of protocols allowing routine acceptance of homozygous genotypes after as few as three replicates may lead to unanticipated errors when applied to low-concentration DNAs.
许多分子生态学研究依赖于对大量低数量 DNA 提取物进行基因分型，这些提取物来自非侵入性或博物馆标本。为了克服低扩增成功率和避免等位基因丢失和假等位基因等基因分型错误，通常对每个样本使用多个聚合酶链反应 (PCR) 重复。最近，引入了两步多重程序，大大提高了基因分型的成功率和效率。然而，对于适当控制错误所需的复制量仍然存在争议。在这里，我们描述了使用两步多重 PCR 程序的方法，该方法允许使用至少 19 个不同的微卫星基因座进行快速基因分型。我们将这种方法应用于来自西部黑猩猩、西部大猩猩、山地大猩猩和黑白疣猴粪便样本的定量非侵入性 DNA，以及来自博物馆的 ~100 年历史的大猩猩牙齿的 DNA。对超过 45000 个 PCR 的分析显示，使用粪便 DNA 的成功率>90%，使用博物馆标本 DNA 的成功率为 74%。与使用传统单重 PCR 协议获得的结果相比，平均等位基因丢失率大大降低，并且可以使用低 (<25 pg) 量的模板 DNA 进行可靠的基因分型。然而，当使用最低浓度的 DNA(<50 pg/反应)时，需要四到五个重复的明显纯合结果，以避免等位基因丢失，这表明使用允许在仅三个重复后常规接受纯合基因型的协议可能会导致在应用于低浓度 DNA 时出现意外错误。
