M. Arandjelovic and K. Guschanski contributed equally to this work.
Two-step multiplex polymerase chain reaction improves the speed and accuracy of genotyping using DNA from noninvasive and museum samples
Article first published online: 20 OCT 2008
© 2008 The Authors. Journal compilation © 2008 Blackwell Publishing Ltd
Molecular Ecology Resources
Volume 9, Issue 1, pages 28–36, January 2009
How to Cite
ARANDJELOVIC, M., GUSCHANSKI, K., SCHUBERT, G., HARRIS, T. R., THALMANN, O., SIEDEL, H. and VIGILANT, L. (2009), Two-step multiplex polymerase chain reaction improves the speed and accuracy of genotyping using DNA from noninvasive and museum samples. Molecular Ecology Resources, 9: 28–36. doi: 10.1111/j.1755-0998.2008.02387.x
- Issue published online: 22 JAN 2009
- Article first published online: 20 OCT 2008
- Received 2 April 2008; revision accepted 29 May 2008
- black and white colobus;
- genotyping errors;
- low-quantity DNA;
- multiplex pre-amplification method
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.