Recursive DNA assembly using protected oligonucleotide duplex assisted cloning (PODAC)
- Author
- Bob Van Hove, Chiara Guidi (UGent) , Lien De Wannemaeker (UGent) , Jo Maertens (UGent) and Marjan De Mey (UGent)
- Organization
- Abstract
- A problem rarely tackled by current DNA assembly methods is the issue of cloning additional parts into an already assembled construct. Costly PCR workflows are often hindered by repeated sequences, and restriction based strategies impose design constraints for each enzyme used. Here we present Protected Oligonucleotide Duplex Assisted Cloning (PODAC), a novel technique that makes use of an oligonucleotide duplex for iterative Golden Gate cloning using only one restriction enzyme. Methylated bases confer protection from digestion during the assembly reaction and are removed during replication in vivo, unveiling a new cloning site in the process. We used this method to efficiently and accurately assemble a biosynthetic pathway and demonstrated its robustness toward sequence repeats by constructing artificial CRISPR arrays. As PODAC is readily amenable to standardization, it would make a useful addition to the synthetic biology toolkit.
- Keywords
- CRISPR, DNA assembly, Golden Gate cloning, molecular cloning, pathway assembly, recursive cloning, SYNTHETIC BIOLOGY, VIOLACEIN, BIOSYNTHESIS, PATHWAYS, CIRCUITS, STANDARD, PROVIDES, PARTS
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Citation
Please use this url to cite or link to this publication: http://hdl.handle.net/1854/LU-8516711
- MLA
- Van Hove, Bob, et al. “Recursive DNA Assembly Using Protected Oligonucleotide Duplex Assisted Cloning (PODAC).” ACS SYNTHETIC BIOLOGY, vol. 6, no. 6, 2017, pp. 943–49, doi:10.1021/acssynbio.7b00017.
- APA
- Van Hove, B., Guidi, C., De Wannemaeker, L., Maertens, J., & De Mey, M. (2017). Recursive DNA assembly using protected oligonucleotide duplex assisted cloning (PODAC). ACS SYNTHETIC BIOLOGY, 6(6), 943–949. https://doi.org/10.1021/acssynbio.7b00017
- Chicago author-date
- Van Hove, Bob, Chiara Guidi, Lien De Wannemaeker, Jo Maertens, and Marjan De Mey. 2017. “Recursive DNA Assembly Using Protected Oligonucleotide Duplex Assisted Cloning (PODAC).” ACS SYNTHETIC BIOLOGY 6 (6): 943–49. https://doi.org/10.1021/acssynbio.7b00017.
- Chicago author-date (all authors)
- Van Hove, Bob, Chiara Guidi, Lien De Wannemaeker, Jo Maertens, and Marjan De Mey. 2017. “Recursive DNA Assembly Using Protected Oligonucleotide Duplex Assisted Cloning (PODAC).” ACS SYNTHETIC BIOLOGY 6 (6): 943–949. doi:10.1021/acssynbio.7b00017.
- Vancouver
- 1.Van Hove B, Guidi C, De Wannemaeker L, Maertens J, De Mey M. Recursive DNA assembly using protected oligonucleotide duplex assisted cloning (PODAC). ACS SYNTHETIC BIOLOGY. 2017;6(6):943–9.
- IEEE
- [1]B. Van Hove, C. Guidi, L. De Wannemaeker, J. Maertens, and M. De Mey, “Recursive DNA assembly using protected oligonucleotide duplex assisted cloning (PODAC),” ACS SYNTHETIC BIOLOGY, vol. 6, no. 6, pp. 943–949, 2017.
@article{8516711,
abstract = {{A problem rarely tackled by current DNA assembly methods is the issue of cloning additional parts into an already assembled construct. Costly PCR workflows are often hindered by repeated sequences, and restriction based strategies impose design constraints for each enzyme used. Here we present Protected Oligonucleotide Duplex Assisted Cloning (PODAC), a novel technique that makes use of an oligonucleotide duplex for iterative Golden Gate cloning using only one restriction enzyme. Methylated bases confer protection from digestion during the assembly reaction and are removed during replication in vivo, unveiling a new cloning site in the process. We used this method to efficiently and accurately assemble a biosynthetic pathway and demonstrated its robustness toward sequence repeats by constructing artificial CRISPR arrays. As PODAC is readily amenable to standardization, it would make a useful addition to the synthetic biology toolkit.}},
author = {{Van Hove, Bob and Guidi, Chiara and De Wannemaeker, Lien and Maertens, Jo and De Mey, Marjan}},
issn = {{2161-5063}},
journal = {{ACS SYNTHETIC BIOLOGY}},
keywords = {{CRISPR,DNA assembly,Golden Gate cloning,molecular cloning,pathway assembly,recursive cloning,SYNTHETIC BIOLOGY,VIOLACEIN,BIOSYNTHESIS,PATHWAYS,CIRCUITS,STANDARD,PROVIDES,PARTS}},
language = {{eng}},
number = {{6}},
pages = {{943--949}},
title = {{Recursive DNA assembly using protected oligonucleotide duplex assisted cloning (PODAC)}},
url = {{http://doi.org/10.1021/acssynbio.7b00017}},
volume = {{6}},
year = {{2017}},
}
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