Keep the DNA rolling: Multiple Displacement Amplification of archival plant DNA extracts

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Abstract:

Insufficient reserves of genomic DNA can hamper molecular phylogenetic analysis. High-throughput genetic techniques that require relatively large amounts of DNA, the difficulty in obtaining samples of taxa from remote regions, and re-sampling of limited archival DNA by repeated phylogenetic surveys can often limit the DNA available for study. To provide a possible solution to this problem, we applied Multiple Displacement Amplification (MDA) to eight archival genomic DNA extracts. The performance of MDA-treated DNA versus untreated genomic extract was evaluated by PCR amplification of three common phylogenetic markers (psbB, nad7, ITS) across a dilution series. Generally, amplification of all three genetic markers from the MDA-treated DNA dilutions was greater than from equivalent dilutions of untreated genomic template. These results indicate that genes from all three plant genomes were amplified and that copies of the target genes psbB, nad7, and ITS were substantially increased during the MDA procedure. Sequencing of the psbB, nad7, and ITS PCR products from both the MDA-treated DNA and the untreated template was used to assess the fidelity of the MDA procedure. Sequences from the MDA-treated DNA and the untreated genomic template differed by 1.2 × 10–4%, which is within the margin of Taq error. These findings emphasize the significance of Multiple Displacement Amplification for optimization of weak PCR, maintenance of depleted genetic stocks, increasing density of taxon sampling, and improving consistency between different phylogenetic analyses.

Keywords: CARYOPHYLLALES; DNA BANK; PLANT DNA EXTRACTION; RCA; ROLLING CIRCLE AMPLIFICATION; WHOLE GENOME AMPLIFICATION

Document Type: Research Article

Affiliations: 1: Department of Botany, University of Florida, Gainesville, Florida 32611, U.S.A.;, Email: sbrockington@flmnh.ufl.edu 2: Florida Museum of Natural History, University of Florida, Gainesville, Florida 32611, U.S.A. 3: Horticulture and Landscape Architecture, Washington State University, Pullman, Washington 99164, U.S.A. 4: Department of Botany, University of Florida, Gainesville, Florida 32611, U.S.A.

Publication date: August 1, 2008

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