skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Cloning should be simple: Escherichia coli DH5α-mediated assembly of multiple DNA fragments with short end homologies

Abstract

Numerous DNA assembly technologies exist for generating plasmids for biological studies. Many procedures require complex in vitro or in vivo assembly reactions followed by plasmid propagation in recombination-impaired Escherichia coli strains such as DH5α, which are optimal for stable amplification of the DNA materials. Here we show that despite its utility as a cloning strain, DH5α retains sufficient recombinase activity to assemble up to six doublestranded DNA fragments ranging in size from 150 bp to at least 7 kb into plasmids in vivo. This process also requires surprisingly small amounts of DNA, potentially obviating the need for upstream assembly processes associated with most common applications of DNA assembly. In addition, we demonstrate the application of this process in cloning of various DNA fragments including synthetic genes, preparation of knockout constructs, and incorporation of guide RNA sequences in constructs for clustered regularly interspaced short palindromic repeats (CRISPR) genome editing. This consolidated process for assembly and amplification in a widely available strain of E. coli may enable productivity gain across disciplines involving recombinant DNA work.

Authors:
 [1];  [2];  [2];  [1];  [3]
  1. J. Craig Venter Institute, La Jolla, CA (United States)
  2. Cornell Univ., Ithaca, NY (United States)
  3. Imperial College, London (United Kingdom)
Publication Date:
Research Org.:
J. Craig Venter Institute Inc., La Jolla, CA (United States); Cornell Univ., Ithaca, NY (United States)
Sponsoring Org.:
USDOE Office of Energy Efficiency and Renewable Energy (EERE); USDOE Office of Science (SC)
OSTI Identifier:
1326190
Grant/Contract Number:  
EE0006109; SC0006644
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
PLoS ONE
Additional Journal Information:
Journal Volume: 10; Journal Issue: 9; Journal ID: ISSN 1932-6203
Publisher:
Public Library of Science
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; polymerase chain reaction; DNA cloning; plasmid construction; cloning; DNA recombination; DNA; homologous recombination; sequence assembly tools

Citation Formats

Kostylev, Maxim, Otwell, Anne E., Richardson, Ruth E., Suzuki, Yo, and Isalan, Mark. Cloning should be simple: Escherichia coli DH5α-mediated assembly of multiple DNA fragments with short end homologies. United States: N. p., 2015. Web. doi:10.1371/journal.pone.0137466.
Kostylev, Maxim, Otwell, Anne E., Richardson, Ruth E., Suzuki, Yo, & Isalan, Mark. Cloning should be simple: Escherichia coli DH5α-mediated assembly of multiple DNA fragments with short end homologies. United States. doi:10.1371/journal.pone.0137466.
Kostylev, Maxim, Otwell, Anne E., Richardson, Ruth E., Suzuki, Yo, and Isalan, Mark. Tue . "Cloning should be simple: Escherichia coli DH5α-mediated assembly of multiple DNA fragments with short end homologies". United States. doi:10.1371/journal.pone.0137466. https://www.osti.gov/servlets/purl/1326190.
@article{osti_1326190,
title = {Cloning should be simple: Escherichia coli DH5α-mediated assembly of multiple DNA fragments with short end homologies},
author = {Kostylev, Maxim and Otwell, Anne E. and Richardson, Ruth E. and Suzuki, Yo and Isalan, Mark},
abstractNote = {Numerous DNA assembly technologies exist for generating plasmids for biological studies. Many procedures require complex in vitro or in vivo assembly reactions followed by plasmid propagation in recombination-impaired Escherichia coli strains such as DH5α, which are optimal for stable amplification of the DNA materials. Here we show that despite its utility as a cloning strain, DH5α retains sufficient recombinase activity to assemble up to six doublestranded DNA fragments ranging in size from 150 bp to at least 7 kb into plasmids in vivo. This process also requires surprisingly small amounts of DNA, potentially obviating the need for upstream assembly processes associated with most common applications of DNA assembly. In addition, we demonstrate the application of this process in cloning of various DNA fragments including synthetic genes, preparation of knockout constructs, and incorporation of guide RNA sequences in constructs for clustered regularly interspaced short palindromic repeats (CRISPR) genome editing. This consolidated process for assembly and amplification in a widely available strain of E. coli may enable productivity gain across disciplines involving recombinant DNA work.},
doi = {10.1371/journal.pone.0137466},
journal = {PLoS ONE},
number = 9,
volume = 10,
place = {United States},
year = {Tue Sep 08 00:00:00 EDT 2015},
month = {Tue Sep 08 00:00:00 EDT 2015}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Citation Metrics:
Cited by: 11 works
Citation information provided by
Web of Science

Save / Share:

Works referenced in this record:

Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension
journal, April 1989


An efficient recombination system for chromosome engineering in Escherichia coli
journal, May 2000

  • Yu, D.; Ellis, H. M.; Lee, E-C.
  • Proceedings of the National Academy of Sciences, Vol. 97, Issue 11, p. 5978-5983
  • DOI: 10.1073/pnas.100127597

Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems
journal, March 2013

  • DiCarlo, James E.; Norville, Julie E.; Mali, Prashant
  • Nucleic Acids Research, Vol. 41, Issue 7, p. 4336-4343
  • DOI: 10.1093/nar/gkt135