Two-step multiplex polymerase chain reaction improves the speed and accuracy of genotyping using DNA from noninvasive and museum samples
Two-step multiplex polymerase chain reaction improves the speed and accuracy of genotyping using DNA from noninvasive and museum samples
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DOI：
10.1111/j.1755-0998.2008.02387.x
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发表时间：
2009-01-01
影响因子：
7.7
通讯作者：
Vigilant, L.
中科院分区：
文献类型：
--
作者：
Arandjelovic, M.;Guschanski, K.;Vigilant, L.
分子生态学的许多研究依赖于对来自非侵入性或博物馆标本的大量低量DNA提取物进行基因分型。为了克服低扩增成功率和避免基因分型错误，如等位基因缺失和假等位基因，通常使用每个样品的多个聚合酶链反应（PCR）重复。最近，两步多重程序已被引入，这大大提高了基因分型的成功率和效率。然而，关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，允许使用至少19个不同的微卫星位点的快速基因分型。我们将这种方法应用于定量的西部黑猩猩，西部大猩猩，山地大猩猩和黑色和白色疣猴粪便样本的非侵入性DNA，以及来自博物馆的100岁大猩猩牙齿的DNA。对超过45000个PCR的分析显示，使用粪便DNA的平均成功率> 90%，使用博物馆标本DNA的平均成功率为74%。与使用常规单重PCR方案获得的那些相比，平均等位基因缺失率显著降低，并且使用低（< 25 pg）量的模板DNA进行可靠的基因分型是可能的。然而，当使用最低浓度的DNA（< 50 pg/反应）时，需要四到五次重复的明显纯合的结果以避免等位基因丢失，这表明当应用于低浓度DNA时，使用允许在少至三次重复后常规接受纯合基因型的方案可能导致意外的错误。
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.