In vivo cloning of up to 16 kb plasmids in E. coli is as simple as PCR
The precise assembly of defined DNA sequences into plasmids is an essential task in bioscience research. While a number of molecular cloning techniques have been developed, many methods require specialized expensive reagents or laborious experimental procedure. Not surprisingly, conventional cloning...
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description | The precise assembly of defined DNA sequences into plasmids is an essential task in bioscience research. While a number of molecular cloning techniques have been developed, many methods require specialized expensive reagents or laborious experimental procedure. Not surprisingly, conventional cloning techniques based on restriction digestion and ligation are still commonly used in routine DNA cloning. Here, we describe a simple, fast, and economical cloning method based on RecA- and RecET-independent in vivo recombination of DNA fragments with overlapping ends using E. coli. All DNA fragments were prepared by a 2-consecutive PCR procedure with Q5 DNA polymerase and used directly for transformation resulting in 95% cloning accuracy and zero background from parental template plasmids. Quantitative relationships were established between cloning efficiency and three factors-the length of overlapping nucleotides, the number of DNA fragments, and the size of target plasmids-which can provide general guidance for selecting in vivo cloning parameters. The method may be used to accurately assemble up to 5 DNA fragments with 25 nt overlapping ends into relatively small plasmids, and 3 DNA fragments into plasmids up to 16 kb in size. The whole cloning procedure may be completed within 2 days by a researcher with little training in cloning. The combination of high accuracy and zero background eliminates the need for screening a large number of colonies. The method requires no enzymes other than Q5 DNA polymerase, has no sequence restriction, is highly reliable, and represents one of the simplest, fastest, and cheapest cloning techniques available. Our method is particularly suitable for common cloning tasks in the lab where the primary goal is to quickly generate a plasmid with a pre-defined sequence at low costs. |
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While a number of molecular cloning techniques have been developed, many methods require specialized expensive reagents or laborious experimental procedure. Not surprisingly, conventional cloning techniques based on restriction digestion and ligation are still commonly used in routine DNA cloning. Here, we describe a simple, fast, and economical cloning method based on RecA- and RecET-independent in vivo recombination of DNA fragments with overlapping ends using E. coli. All DNA fragments were prepared by a 2-consecutive PCR procedure with Q5 DNA polymerase and used directly for transformation resulting in 95% cloning accuracy and zero background from parental template plasmids. Quantitative relationships were established between cloning efficiency and three factors-the length of overlapping nucleotides, the number of DNA fragments, and the size of target plasmids-which can provide general guidance for selecting in vivo cloning parameters. The method may be used to accurately assemble up to 5 DNA fragments with 25 nt overlapping ends into relatively small plasmids, and 3 DNA fragments into plasmids up to 16 kb in size. The whole cloning procedure may be completed within 2 days by a researcher with little training in cloning. The combination of high accuracy and zero background eliminates the need for screening a large number of colonies. The method requires no enzymes other than Q5 DNA polymerase, has no sequence restriction, is highly reliable, and represents one of the simplest, fastest, and cheapest cloning techniques available. Our method is particularly suitable for common cloning tasks in the lab where the primary goal is to quickly generate a plasmid with a pre-defined sequence at low costs.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0183974</identifier><identifier>PMID: 28837659</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Biochemistry ; Biology and Life Sciences ; Chromosomes ; Cloning ; Cloning, Molecular ; Colonies ; Deoxyribonucleic acid ; Digestion ; DNA ; DNA polymerase ; DNA sequencing ; DNA-directed DNA polymerase ; E coli ; Efficiency ; Enzymes ; Escherichia coli ; Escherichia coli - genetics ; Fragmentation ; Fragments ; Gene sequencing ; In vivo methods and tests ; Kinases ; Methods ; Mutagenesis ; Nucleotide sequence ; Nucleotides ; Plasmids ; Polymerase chain reaction ; Polymerase Chain Reaction - methods ; Reagents ; RecA protein ; Recombination ; Research and Analysis Methods ; Studies ; Transformation</subject><ispartof>PloS one, 2017-08, Vol.12 (8), p.e0183974-e0183974</ispartof><rights>COPYRIGHT 2017 Public Library of Science</rights><rights>2017 Huang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2017 Huang et al 2017 Huang et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c758t-4ab87224d3a29090fdf7ae1aac8e014afb8b21689ce6de1d00db66edbe8cfc9e3</citedby><cites>FETCH-LOGICAL-c758t-4ab87224d3a29090fdf7ae1aac8e014afb8b21689ce6de1d00db66edbe8cfc9e3</cites><orcidid>0000-0001-8185-7097</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5570364/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5570364/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,724,777,781,861,882,2096,2915,23847,27905,27906,53772,53774,79349,79350</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/28837659$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Isalan, Mark</contributor><creatorcontrib>Huang, Faqing</creatorcontrib><creatorcontrib>Spangler, Joseph Rankin</creatorcontrib><creatorcontrib>Huang, Allen Yang</creatorcontrib><title>In vivo cloning of up to 16 kb plasmids in E. coli is as simple as PCR</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>The precise assembly of defined DNA sequences into plasmids is an essential task in bioscience research. While a number of molecular cloning techniques have been developed, many methods require specialized expensive reagents or laborious experimental procedure. Not surprisingly, conventional cloning techniques based on restriction digestion and ligation are still commonly used in routine DNA cloning. Here, we describe a simple, fast, and economical cloning method based on RecA- and RecET-independent in vivo recombination of DNA fragments with overlapping ends using E. coli. All DNA fragments were prepared by a 2-consecutive PCR procedure with Q5 DNA polymerase and used directly for transformation resulting in 95% cloning accuracy and zero background from parental template plasmids. Quantitative relationships were established between cloning efficiency and three factors-the length of overlapping nucleotides, the number of DNA fragments, and the size of target plasmids-which can provide general guidance for selecting in vivo cloning parameters. The method may be used to accurately assemble up to 5 DNA fragments with 25 nt overlapping ends into relatively small plasmids, and 3 DNA fragments into plasmids up to 16 kb in size. The whole cloning procedure may be completed within 2 days by a researcher with little training in cloning. The combination of high accuracy and zero background eliminates the need for screening a large number of colonies. The method requires no enzymes other than Q5 DNA polymerase, has no sequence restriction, is highly reliable, and represents one of the simplest, fastest, and cheapest cloning techniques available. Our method is particularly suitable for common cloning tasks in the lab where the primary goal is to quickly generate a plasmid with a pre-defined sequence at low costs.</description><subject>Biochemistry</subject><subject>Biology and Life Sciences</subject><subject>Chromosomes</subject><subject>Cloning</subject><subject>Cloning, Molecular</subject><subject>Colonies</subject><subject>Deoxyribonucleic acid</subject><subject>Digestion</subject><subject>DNA</subject><subject>DNA polymerase</subject><subject>DNA sequencing</subject><subject>DNA-directed DNA polymerase</subject><subject>E coli</subject><subject>Efficiency</subject><subject>Enzymes</subject><subject>Escherichia coli</subject><subject>Escherichia coli - genetics</subject><subject>Fragmentation</subject><subject>Fragments</subject><subject>Gene sequencing</subject><subject>In vivo methods and tests</subject><subject>Kinases</subject><subject>Methods</subject><subject>Mutagenesis</subject><subject>Nucleotide sequence</subject><subject>Nucleotides</subject><subject>Plasmids</subject><subject>Polymerase chain reaction</subject><subject>Polymerase Chain Reaction - 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genetics</topic><topic>Fragmentation</topic><topic>Fragments</topic><topic>Gene sequencing</topic><topic>In vivo methods and tests</topic><topic>Kinases</topic><topic>Methods</topic><topic>Mutagenesis</topic><topic>Nucleotide sequence</topic><topic>Nucleotides</topic><topic>Plasmids</topic><topic>Polymerase chain reaction</topic><topic>Polymerase Chain Reaction - methods</topic><topic>Reagents</topic><topic>RecA protein</topic><topic>Recombination</topic><topic>Research and Analysis Methods</topic><topic>Studies</topic><topic>Transformation</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Huang, Faqing</creatorcontrib><creatorcontrib>Spangler, Joseph Rankin</creatorcontrib><creatorcontrib>Huang, Allen Yang</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Gale In Context: Opposing Viewpoints</collection><collection>Gale In Context: Science</collection><collection>ProQuest Central (Corporate)</collection><collection>Animal Behavior Abstracts</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Biotechnology Research Abstracts</collection><collection>Nursing & Allied Health Database</collection><collection>Ecology Abstracts</collection><collection>Entomology Abstracts (Full archive)</collection><collection>Immunology Abstracts</collection><collection>Meteorological & Geoastrophysical Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Agricultural Science Collection</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Medical Database (Alumni Edition)</collection><collection>ProQuest Pharma Collection</collection><collection>Public Health Database</collection><collection>Technology Research Database</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Technology Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>Materials Science & Engineering Collection</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest One Sustainability</collection><collection>ProQuest Central UK/Ireland</collection><collection>Advanced Technologies & Aerospace Collection</collection><collection>Agricultural & Environmental Science Collection</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Technology Collection</collection><collection>Natural Science Collection</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ProQuest One Community College</collection><collection>ProQuest Materials Science Collection</collection><collection>ProQuest Central Korea</collection><collection>Engineering Research Database</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>Materials Science Database</collection><collection>Nursing & Allied Health Database (Alumni Edition)</collection><collection>Meteorological & Geoastrophysical Abstracts - 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Academic</collection><collection>PubMed Central (Full Participant titles)</collection><collection>DOAJ Directory of Open Access Journals</collection><jtitle>PloS one</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Huang, Faqing</au><au>Spangler, Joseph Rankin</au><au>Huang, Allen Yang</au><au>Isalan, Mark</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>In vivo cloning of up to 16 kb plasmids in E. coli is as simple as PCR</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2017-08-24</date><risdate>2017</risdate><volume>12</volume><issue>8</issue><spage>e0183974</spage><epage>e0183974</epage><pages>e0183974-e0183974</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><abstract>The precise assembly of defined DNA sequences into plasmids is an essential task in bioscience research. While a number of molecular cloning techniques have been developed, many methods require specialized expensive reagents or laborious experimental procedure. Not surprisingly, conventional cloning techniques based on restriction digestion and ligation are still commonly used in routine DNA cloning. Here, we describe a simple, fast, and economical cloning method based on RecA- and RecET-independent in vivo recombination of DNA fragments with overlapping ends using E. coli. All DNA fragments were prepared by a 2-consecutive PCR procedure with Q5 DNA polymerase and used directly for transformation resulting in 95% cloning accuracy and zero background from parental template plasmids. Quantitative relationships were established between cloning efficiency and three factors-the length of overlapping nucleotides, the number of DNA fragments, and the size of target plasmids-which can provide general guidance for selecting in vivo cloning parameters. The method may be used to accurately assemble up to 5 DNA fragments with 25 nt overlapping ends into relatively small plasmids, and 3 DNA fragments into plasmids up to 16 kb in size. The whole cloning procedure may be completed within 2 days by a researcher with little training in cloning. The combination of high accuracy and zero background eliminates the need for screening a large number of colonies. The method requires no enzymes other than Q5 DNA polymerase, has no sequence restriction, is highly reliable, and represents one of the simplest, fastest, and cheapest cloning techniques available. Our method is particularly suitable for common cloning tasks in the lab where the primary goal is to quickly generate a plasmid with a pre-defined sequence at low costs.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>28837659</pmid><doi>10.1371/journal.pone.0183974</doi><tpages>e0183974</tpages><orcidid>https://orcid.org/0000-0001-8185-7097</orcidid><oa>free_for_read</oa></addata></record> |
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subjects | Biochemistry Biology and Life Sciences Chromosomes Cloning Cloning, Molecular Colonies Deoxyribonucleic acid Digestion DNA DNA polymerase DNA sequencing DNA-directed DNA polymerase E coli Efficiency Enzymes Escherichia coli Escherichia coli - genetics Fragmentation Fragments Gene sequencing In vivo methods and tests Kinases Methods Mutagenesis Nucleotide sequence Nucleotides Plasmids Polymerase chain reaction Polymerase Chain Reaction - methods Reagents RecA protein Recombination Research and Analysis Methods Studies Transformation |
title | In vivo cloning of up to 16 kb plasmids in E. coli is as simple as PCR |
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