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      Evaluation of Five Methods for Total DNA Extraction from Western Corn Rootworm Beetles

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          Abstract

          Background

          DNA extraction is a routine step in many insect molecular studies. A variety of methods have been used to isolate DNA molecules from insects, and many commercial kits are available. Extraction methods need to be evaluated for their efficiency, cost, and side effects such as DNA degradation during extraction.

          Methodology/Principal Findings

          From individual western corn rootworm beetles, Diabrotica virgifera virgifera, DNA extractions by the SDS method, CTAB method, DNAzol® reagent, Puregene® solutions and DNeasy® column were compared in terms of DNA quantity and quality, cost of materials, and time consumed. Although all five methods resulted in acceptable DNA concentrations and absorbance ratios, the SDS and CTAB methods resulted in higher DNA yield (ng DNA vs. mg tissue) at much lower cost and less degradation as revealed on agarose gels. The DNeasy® kit was most time-efficient but was the costliest among the methods tested. The effects of ethanol volume, temperature and incubation time on precipitation of DNA were also investigated. The DNA samples obtained by the five methods were tested in PCR for six microsatellites located in various positions of the beetle's genome, and all samples showed successful amplifications.

          Conclusion/Significance

          These evaluations provide a guide for choosing methods of DNA extraction from western corn rootworm beetles based on expected DNA yield and quality, extraction time, cost, and waste control. The extraction conditions for this mid-size insect were optimized. The DNA extracted by the five methods was suitable for further molecular applications such as PCR and sequencing by synthesis.

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          Most cited references 9

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          Universal and rapid salt-extraction of high quality genomic DNA for PCR-based techniques.

           S. Aljanabi (1997)
          A very simple, fast, universally applicable and reproducible method to extract high quality megabase genomic DNA from different organisms is described. We applied the same method to extract high quality complex genomic DNA from different tissues (wheat, barley, potato, beans, pear and almond leaves as well as fungi, insects and shrimps' fresh tissue) without any modification. The method does not require expensive and environmentally hazardous reagents and equipment. It can be performed even in low technology laboratories. The amount of tissue required by this method is approximately 50-100 mg. The quantity and the quality of the DNA extracted by this method is high enough to perform hundreds of PCR-based reactions and also to be used in other DNA manipulation techniques such as restriction digestion, Southern blot and cloning.
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            DNA Extraction from Dry Museum Beetles without Conferring External Morphological Damage

            Background A large number of dry-preserved insect specimens exist in collections around the world that might be useful for genetic analyses. However, until now, the recovery of nucleic acids from such specimens has involved at least the partial destruction of the specimen. This is clearly undesirable when dealing with rare species or otherwise important specimens, such as type specimens. Methodology We describe a method for the extraction of PCR-amplifiable mitochondrial and nuclear DNA from dry insects without causing external morphological damage. Using PCR to amplify ≈220 bp of the mitochondrial gene cytochrome c oxidase I, and 250–345 bp fragments of the multi-copy, nuclear 28s ribosomal DNA gene, we demonstrate the efficacy of this method on beetles collected up to 50 years ago. Conclusions This method offers a means of obtaining useful genetic information from rare insects without conferring external morphological damage.
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              Mitochondrial DNA in the bark weevils: size, structure and heteroplasmy.

              Mitochondrial DNA of higher animals has been described as an example of extreme efficiency in genome structure and function. Where exceptionally large size molecules have been found (greater than 20 kb), most have occurred as rare variants within a species, suggesting that these variants arise infrequently and do not persist for long periods in evolutionary time. In contrast, all individuals of at least three species of bark weevil (Curculionidae: Pissodes) possess a mitochondrial genome of unusually large size (30-36 kb). The molecule owes its large size to a dramatically enlarged A + T-rich region (9-13 kb). Gene content and order outside of this region appear to be identical to that found in Drosophila. A series of 0.8-2.0-kb repeated sequences occur adjacent to the large A + T rich region and have perhaps played a role in the generation of the large size as well as an unprecedented frequency of size variant heteroplasmy. Every weevil sampled in all three species (n = 219) exhibits anywhere from two to five distinct size classes of mtDNA. The persistence of this large amount of size polymorphism through two speciation events combined with the abundant size variation within individuals suggests that these molecules may not be subject to strong selection for small overall size and efficiency of replication. This pattern of variation contrasts strongly with the conservation of gene content and arrangement in the coding region of the molecule.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2010
                13 August 2010
                : 5
                : 8
                Affiliations
                Department of Entomology, University of Nebraska-Lincoln, Lincoln, Nebraska, United States of America
                Institute of Evolutionary Biology (CSIC-UPF), Spain
                Author notes

                Conceived and designed the experiments: HC BDS. Performed the experiments: HC MR SYT HW. Analyzed the data: HC. Contributed reagents/materials/analysis tools: HC MR SYT HW. Wrote the paper: HC BDS.

                Article
                10-PONE-RA-19367R1
                10.1371/journal.pone.0011963
                2921343
                20730102
                Chen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                Page count
                Pages: 6
                Categories
                Research Article
                Biochemistry
                Biotechnology
                Genetics and Genomics
                Molecular Biology

                Uncategorized

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