The problems and promise of DNA barcodes for species diagnosis of primate biomaterials - PubMed (original) (raw)
Comparative Study
The problems and promise of DNA barcodes for species diagnosis of primate biomaterials
Joseph G Lorenz et al. Philos Trans R Soc Lond B Biol Sci. 2005.
Abstract
The Integrated Primate Biomaterials and Information Resource (www.IPBIR.org) provides essential research reagents to the scientific community by establishing, verifying, maintaining, and distributing DNA and RNA derived from primate cell cultures. The IPBIR uses mitochondrial cytochrome c oxidase subunit I sequences to verify the identity of samples for quality control purposes in the accession, cell culture, DNA extraction processes and prior to shipping to end users. As a result, IPBIR is accumulating a database of 'DNA barcodes' for many species of primates. However, this quality control process is complicated by taxon specific patterns of 'universal primer' failure, as well as the amplification or co-amplification of nuclear pseudogenes of mitochondrial origins. To overcome these difficulties, taxon specific primers have been developed, and reverse transcriptase PCR is utilized to exclude these extraneous sequences from amplification. DNA barcoding of primates has applications to conservation and law enforcement. Depositing barcode sequences in a public database, along with primer sequences, trace files and associated quality scores, makes this species identification technique widely accessible. Reference DNA barcode sequences should be derived from, and linked to, specimens of known provenance in web-accessible collections in order to validate this system of molecular diagnostics.
Figures
Figure 1
Results of rtPCR performed on RNA extracted from Papio anubis liver. Lanes 1 & 13; Msp I digested pBR322 ladder. Lane 2; PGK positive control. Lane 3; PGK DNA control (no RNA). Lane 4; amplification of genomic DNA using primer set1. Lane 5; rtPCR with primer set 1. Lane 6; ‘mock’ rtPCR using AmpliTaq instead of reverse transcriptase and primer set 1. Lane 7; amplification of genomic DNA using primer set2. Lane 8; rtPCR with primer set 2. Lane 9; ‘mock’ rtPCR using AmpliTaq instead of reverse transcriptase and primer set 2. Lane 10; amplification of genomic DNA using primer set 3. Lane 11; rtPCR with primer set 3. Lane 12; ‘mock’ rtPCR using AmpliTaq instead of reverse transcriptase and primer set 3.
Figure 2
Bootstrapped neighbour-joining tree calculated from Kimura 2p distances and based on 1000 replicates. The number in parentheses indicates the number of samples that share identical sequences at that position; #, indicates the position of cox1 sequences obtained from GenBank.
Figure 3
Mean pairwise differences (Kimura 2p) at various taxonomic levels.
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