The DOI matches its Crossref record; 2 of the 2 references it cites were found in a registry or an index.
Cloning Should Be Simple: Escherichia coli DH5α-Mediated Assembly of Multiple DNA Fragments with Short End Homologies
Open accessAbstract
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 double-stranded 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. 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.
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Citations
Cites
CRISPR-Cas systems for editing, regulating and targeting genomes
Sander, J. D., & Joung, J. K. (2014) · Nature Biotechnology · doi:10.1038/nbt.2842 · Cited by 3,153 · cited 1×
“The most studied and developed system uses CRISPRassociated protein 9 (Cas9) from Streptococcus pyogenes to create a double-stranded DNA break at a specified sequence [33].”
Knocking out multigene redundancies via cycles of sexual assortment and fluorescence selection
Suzuki, Y., Onge, R. P. S., Mani, R., King, O. D., Heilbut, A., Labunskyy, V. M., Chen, W., Pham, L., Zhang, L. V., Tong, A. H. Y., et al. (2011) · Nature Methods · doi:10.1038/nmeth.1550 · Cited by 78
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Cited by
Xia, Y., Li, K., Li, J., Wang, T., Gu, L., & Xun, L. (2019) · Nucleic Acids Research · doi:10.1093/nar/gky1169 · Cited by 317
Kulcsár, P. I., Tálas, A., Tóth, E., Nyeste, A., Ligeti, Z., Welker, Z., & Welker, E. (2020) · Nature Communications · doi:10.1038/s41467-020-15021-5 · Cited by 45
Genetic requirements for cell division in a genomically minimal cell
Pelletier, J. F., Sun, L., Wise, K. S., Assad-Garcia, N., Karas, B. J., Deerinck, T. J., Ellisman, M. H., Mershin, A., Gershenfeld, N., Chuang, R.-Y., et al. (2020) · bioRxiv / Cell · doi:10.1101/2020.10.07.326892 · Cited by 148
Metadata
- Authors
- Kostylev, M., Otwell, A. E., Richardson, R. E., & Suzuki, Y.
- Venue
- PLOS ONE
- Publisher
- Public Library of Science (PLoS)
- Published
- 2015-09-08
- Volume
- 10
- Issue
- 9
- Pages
- e0137466
- ISSN
- 1932-6203 (ISSN-L)
- OpenAlex
- W2123781955
- Cited by
- 162
- References
- 2
- FWCI
- 2.83
- Type
- journal article
- Language
- en
- Open access
- gold
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- Kostylev, M., Otwell, A. E., Richardson, R. E., & Suzuki, Y. (2015). Cloning Should Be Simple: Escherichia coli DH5α-Mediated Assembly of Multiple DNA Fragments with Short End Homologies. PLOS ONE. https://doi.org/10.1371/journal.pone.0137466
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