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Removing PCR for the elimination of undesired DNA fragments cycle by cycle

A novel removing polymerase chain reaction (R-PCR) technique was developed, which can eliminate undesired genes, cycle by cycle, with efficiencies of 60.9% (cDNAs), 73.6% (genomic DNAs), and ~ 100% (four DNA fragments were tested). Major components of the R-PCR include drivers, a thermostable restri...

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Autores principales: Huan, Jiaojiao, Wan, Kangkang, Liu, Yunjun, Dong, Wubei, Wang, Guoying
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3725479/
https://www.ncbi.nlm.nih.gov/pubmed/23892515
http://dx.doi.org/10.1038/srep02303
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author Huan, Jiaojiao
Wan, Kangkang
Liu, Yunjun
Dong, Wubei
Wang, Guoying
author_facet Huan, Jiaojiao
Wan, Kangkang
Liu, Yunjun
Dong, Wubei
Wang, Guoying
author_sort Huan, Jiaojiao
collection PubMed
description A novel removing polymerase chain reaction (R-PCR) technique was developed, which can eliminate undesired genes, cycle by cycle, with efficiencies of 60.9% (cDNAs), 73.6% (genomic DNAs), and ~ 100% (four DNA fragments were tested). Major components of the R-PCR include drivers, a thermostable restriction enzyme - ApeKI, and a poly(dA) adapter with mismatched restriction enzyme recognition sites. Drivers were generated from the undesired genes. In each cycle of R-PCR, drivers anneal to complementary sequences and allow extension by Taq DNA polymerase. Thus, ApeKI restriction sites in the undesired genes are recovered, and adapters of these undesired DNA fragments are removed. Using R-PCR, we isolated maize upregulated defense-responsive genes and Blumeria graminis specialized genes, including key pathogenesis-related effectors. Our results show that after the R-PCR reaction, most undesired genes, including very abundant genes, became undetectable. The R-PCR is an easy and cost-efficient method to eliminate undesired genes and clone desired genes.
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spelling pubmed-37254792013-07-29 Removing PCR for the elimination of undesired DNA fragments cycle by cycle Huan, Jiaojiao Wan, Kangkang Liu, Yunjun Dong, Wubei Wang, Guoying Sci Rep Article A novel removing polymerase chain reaction (R-PCR) technique was developed, which can eliminate undesired genes, cycle by cycle, with efficiencies of 60.9% (cDNAs), 73.6% (genomic DNAs), and ~ 100% (four DNA fragments were tested). Major components of the R-PCR include drivers, a thermostable restriction enzyme - ApeKI, and a poly(dA) adapter with mismatched restriction enzyme recognition sites. Drivers were generated from the undesired genes. In each cycle of R-PCR, drivers anneal to complementary sequences and allow extension by Taq DNA polymerase. Thus, ApeKI restriction sites in the undesired genes are recovered, and adapters of these undesired DNA fragments are removed. Using R-PCR, we isolated maize upregulated defense-responsive genes and Blumeria graminis specialized genes, including key pathogenesis-related effectors. Our results show that after the R-PCR reaction, most undesired genes, including very abundant genes, became undetectable. The R-PCR is an easy and cost-efficient method to eliminate undesired genes and clone desired genes. Nature Publishing Group 2013-07-29 /pmc/articles/PMC3725479/ /pubmed/23892515 http://dx.doi.org/10.1038/srep02303 Text en Copyright © 2013, Macmillan Publishers Limited. All rights reserved http://creativecommons.org/licenses/by-nc-sa/3.0/ This work is licensed under a Creative Commons Attribution-NonCommercial-ShareALike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/
spellingShingle Article
Huan, Jiaojiao
Wan, Kangkang
Liu, Yunjun
Dong, Wubei
Wang, Guoying
Removing PCR for the elimination of undesired DNA fragments cycle by cycle
title Removing PCR for the elimination of undesired DNA fragments cycle by cycle
title_full Removing PCR for the elimination of undesired DNA fragments cycle by cycle
title_fullStr Removing PCR for the elimination of undesired DNA fragments cycle by cycle
title_full_unstemmed Removing PCR for the elimination of undesired DNA fragments cycle by cycle
title_short Removing PCR for the elimination of undesired DNA fragments cycle by cycle
title_sort removing pcr for the elimination of undesired dna fragments cycle by cycle
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3725479/
https://www.ncbi.nlm.nih.gov/pubmed/23892515
http://dx.doi.org/10.1038/srep02303
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