Cargando…

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

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 degradati...

Descripción completa

Detalles Bibliográficos
Autores principales: Chen, Hong, Rangasamy, Murugesan, Tan, Sek Yee, Wang, Haichuan, Siegfried, Blair D.
Formato: Texto
Lenguaje:English
Publicado: Public Library of Science 2010
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2921343/
https://www.ncbi.nlm.nih.gov/pubmed/20730102
http://dx.doi.org/10.1371/journal.pone.0011963
_version_ 1782185376316653568
author Chen, Hong
Rangasamy, Murugesan
Tan, Sek Yee
Wang, Haichuan
Siegfried, Blair D.
author_facet Chen, Hong
Rangasamy, Murugesan
Tan, Sek Yee
Wang, Haichuan
Siegfried, Blair D.
author_sort Chen, Hong
collection PubMed
description 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.
format Text
id pubmed-2921343
institution National Center for Biotechnology Information
language English
publishDate 2010
publisher Public Library of Science
record_format MEDLINE/PubMed
spelling pubmed-29213432010-08-20 Evaluation of Five Methods for Total DNA Extraction from Western Corn Rootworm Beetles Chen, Hong Rangasamy, Murugesan Tan, Sek Yee Wang, Haichuan Siegfried, Blair D. PLoS One Research Article 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. Public Library of Science 2010-08-13 /pmc/articles/PMC2921343/ /pubmed/20730102 http://dx.doi.org/10.1371/journal.pone.0011963 Text en Chen et al. http://creativecommons.org/licenses/by/4.0/ 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 properly credited.
spellingShingle Research Article
Chen, Hong
Rangasamy, Murugesan
Tan, Sek Yee
Wang, Haichuan
Siegfried, Blair D.
Evaluation of Five Methods for Total DNA Extraction from Western Corn Rootworm Beetles
title Evaluation of Five Methods for Total DNA Extraction from Western Corn Rootworm Beetles
title_full Evaluation of Five Methods for Total DNA Extraction from Western Corn Rootworm Beetles
title_fullStr Evaluation of Five Methods for Total DNA Extraction from Western Corn Rootworm Beetles
title_full_unstemmed Evaluation of Five Methods for Total DNA Extraction from Western Corn Rootworm Beetles
title_short Evaluation of Five Methods for Total DNA Extraction from Western Corn Rootworm Beetles
title_sort evaluation of five methods for total dna extraction from western corn rootworm beetles
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2921343/
https://www.ncbi.nlm.nih.gov/pubmed/20730102
http://dx.doi.org/10.1371/journal.pone.0011963
work_keys_str_mv AT chenhong evaluationoffivemethodsfortotaldnaextractionfromwesterncornrootwormbeetles
AT rangasamymurugesan evaluationoffivemethodsfortotaldnaextractionfromwesterncornrootwormbeetles
AT tansekyee evaluationoffivemethodsfortotaldnaextractionfromwesterncornrootwormbeetles
AT wanghaichuan evaluationoffivemethodsfortotaldnaextractionfromwesterncornrootwormbeetles
AT siegfriedblaird evaluationoffivemethodsfortotaldnaextractionfromwesterncornrootwormbeetles