Two-step multiplex PCR improves the speed and accuracy of genotyping using DNA from noninvasive and museum samples

Max Planck Institute for Evolutionary Anthropology, Deutscher Platz 6, D-04103 Leipzig, Germany
Molecular Ecology Resources (Impact Factor: 3.71). 12/2008; 9(1):28 - 36. DOI: 10.1111/j.1755-0998.2008.02387.x
Source: OAI


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.

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Available from: Mimi Arandjelovic
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    • "For each extract, DNA was quantified following the procedures ofMorin et al. (2001). The required number of PCR replicates was then performed to maintain error rates of less than 1 % when scoring homozygotes, as determined byArandjelovic et al. (2009). Heterozygotes were scored by observing both alleles in at least two independent PCRs. "
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    • "The thermocycling conditions were the same as in step 1, except that a primer-specific annealing temperature was used for each singleplex PCR and varied from 55 °C and 60 °C (see Arandjelovic et al., 2009 for details). Four different PCR products were then pooled in each of three different sets of loci, and electrophoresed on an ABI "
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