Highly accurate SNP genotyping from historical and low-quality samples
2007
Morin, Phillip A. | McCarthy, Melissa
Historical and other poor-quality samples are often necessary for population genetics, conservation, and forensics studies. Although there is a long history of using mtDNA from such samples, obtaining and genotyping nuclear loci have been considered difficult and error-prone at best, and impossible at worst. The primary issues are the amount of nuclear DNA available for genotyping, and the degradation of the DNA into small fragments. Single nucleotide polymorphisms offer potential advantages for assaying nuclear variation in historical and poor-quality samples, because the amplified fragments can be very small, varying little or not at all in size between alleles, and can be amplified efficiently by polymerase chain reaction (PCR). We present a method for highly multiplexed PCR of SNP loci, followed by dual-fluorescence genotyping that is very effective for genotyping poor-quality samples, and can potentially use very little template DNA, regardless of the number of loci to be genotyped. We genotyped 19 SNP loci from DNA extracted from modern and historical bowhead whale tissue, bone and baleen samples. The PCR failure rate was < 1.5%, and the genotyping error rate was 0.1% when DNA samples contained > 10 copies/μL of a 51-bp nuclear sequence. Among samples with <= 10 copies/mu L DNA, samples could still be genotyped confidently with appropriate levels of replication from independent multiplex PCRs.
显示更多 [+] 显示较少 [-]