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Suicide polymerase endonuclease restriction, a novel technique for enhancing PCR amplification of minor DNA templates
Year:
2005
Authors :
Minz, Dror
;
.
Volume :
71
Co-Authors:
Green, S.J., Institute of Soil, Water, and Environmental Sciences, Agriculture Research Organization, Volcani Center, Bet-Dagan, Israel, Faculty of Agricultural, Food, and Environmental Quality Sciences, Hebrew University of Jerusalem, Rehovot, Israel, Exobiology Branch, NASA-Ames Research Center, Moffett Field, CA, United States
Minz, D., Institute of Soil, Water, and Environmental Sciences, Agriculture Research Organization, Volcani Center, Bet-Dagan, Israel, Institute of Soil, Water, and Environmental Sciences, Agriculture Research Organization, Volcani Center, P.O. Box 6, Bet-Dagan 50-250, Israel
Facilitators :
From page:
4721
To page:
4727
(
Total pages:
7
)
Abstract:
PCR-based molecular analyses can be hindered by the presence of unwanted or dominant DNA templates that reduce or eliminate detection of alternate templates. We describe here a reaction in which such templates can be exclusively digested by endonuclease restriction, leaving all other DNAs unmodified. After such a modification, the digested template is no longer available for PCR amplification, while nontarget DNAs remain intact and can be amplified. We demonstrate the application of this method and use denaturing gradient gel electrophoresis to ascertain the removal of target DNA templates and the subsequent enhanced amplification of nondigested DNAs. Specifically, plastid 16S rRNA genes were exclusively digested from environmental DNA extracted from plant roots. In addition, pure culture and environmental DNA extracts were spiked with various amounts of genomic DNA extracted from Streptomyces spp., and selective restriction of the Streptomyces 16S rRNA genes via the suicide polymerase endonuclease restriction PCR method was employed to remove the amended DNA. Copyright © 2005, American Society for Microbiology. All Rights Reserved.
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More details
DOI :
10.1128/AEM.71.8.4721-4727.2005
Article number:
Affiliations:
Database:
Scopus
Publication Type:
article
;
.
Language:
English
Editors' remarks:
ID:
20295
Last updated date:
02/03/2022 17:27
Creation date:
16/04/2018 23:35
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Scientific Publication
Suicide polymerase endonuclease restriction, a novel technique for enhancing PCR amplification of minor DNA templates
71
Green, S.J., Institute of Soil, Water, and Environmental Sciences, Agriculture Research Organization, Volcani Center, Bet-Dagan, Israel, Faculty of Agricultural, Food, and Environmental Quality Sciences, Hebrew University of Jerusalem, Rehovot, Israel, Exobiology Branch, NASA-Ames Research Center, Moffett Field, CA, United States
Minz, D., Institute of Soil, Water, and Environmental Sciences, Agriculture Research Organization, Volcani Center, Bet-Dagan, Israel, Institute of Soil, Water, and Environmental Sciences, Agriculture Research Organization, Volcani Center, P.O. Box 6, Bet-Dagan 50-250, Israel
Suicide polymerase endonuclease restriction, a novel technique for enhancing PCR amplification of minor DNA templates
PCR-based molecular analyses can be hindered by the presence of unwanted or dominant DNA templates that reduce or eliminate detection of alternate templates. We describe here a reaction in which such templates can be exclusively digested by endonuclease restriction, leaving all other DNAs unmodified. After such a modification, the digested template is no longer available for PCR amplification, while nontarget DNAs remain intact and can be amplified. We demonstrate the application of this method and use denaturing gradient gel electrophoresis to ascertain the removal of target DNA templates and the subsequent enhanced amplification of nondigested DNAs. Specifically, plastid 16S rRNA genes were exclusively digested from environmental DNA extracted from plant roots. In addition, pure culture and environmental DNA extracts were spiked with various amounts of genomic DNA extracted from Streptomyces spp., and selective restriction of the Streptomyces 16S rRNA genes via the suicide polymerase endonuclease restriction PCR method was employed to remove the amended DNA. Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Scientific Publication
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