Highly efficient multiplex PCR of noninvasive DNA does not require pre‐amplification
Abstract Among the key issues determining success of a study employing molecular genetics tools in wildlife monitoring or research is a large enough set of highly informative genetic markers and a reliable, cost effective method for their analysis. While optimized commercial genotyping kits have bee...
| Published in: | Molecular Ecology Resources |
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| Main Authors: | , , , , |
| Format: | Article in Journal/Newspaper |
| Language: | English |
| Published: |
Wiley
2010
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| Subjects: | |
| Online Access: | http://dx.doi.org/10.1111/j.1755-0998.2009.02780.x https://api.wiley.com/onlinelibrary/tdm/v1/articles/10.1111%2Fj.1755-0998.2009.02780.x https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1755-0998.2009.02780.x https://onlinelibrary.wiley.com/doi/full-xml/10.1111/j.1755-0998.2009.02780.x |
| Summary: | Abstract Among the key issues determining success of a study employing molecular genetics tools in wildlife monitoring or research is a large enough set of highly informative genetic markers and a reliable, cost effective method for their analysis. While optimized commercial genotyping kits have been developed for humans and domestic animals, such protocols are rare in wildlife research. We developed a highly optimized multiplex PCR that genotypes 12 microsatellite loci and a sex determination locus in brown bear ( Ursus arctos ) faecal samples in a single multiplex PCR and a single sequencer run. We used this protocol to genotype 1053 faecal samples of bears from the Dinaric population, and obtained useful genotypes for 88% of the samples, a very high success rate. The new protocol outperformed the multiplex pre‐amplification strategy used in a previous study of 473 faecal samples with a 78.4% success rate. On a subset of 182 samples we directly compared the performance of both approaches, and found no advantage of the multiplex pre‐amplification. While pre‐amplification protocols might still improve PCR success and reliability on a small fraction of low‐quality samples, the higher costs and workload do not justify their use when analysing reasonably fresh non‐invasive material. Moreover, the high number of multiplexed loci in the new protocol makes it comparable to commercially developed genotyping kits developed for domestic animals and humans. |
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