Iterative capped assembly: rapid and scalable synthesis of repeat-module DNA such as TAL effectors from individual monomers
DNA built from modular repeats presents a challenge for gene synthesis. We present a solid surface-based sequential ligation approach, which we refer to as iterative capped assembly (ICA), that adds DNA repeat monomers individually to a growing chain while using hairpin ‘capping’ oligonucleotides to...
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| Format: | Online |
| Language: | English |
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Oxford University Press
2012
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| Online Access: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3424587/ |
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pubmed-34245872012-08-22 Iterative capped assembly: rapid and scalable synthesis of repeat-module DNA such as TAL effectors from individual monomers Briggs, Adrian W. Rios, Xavier Chari, Raj Yang, Luhan Zhang, Feng Mali, Prashant Church, George M. Methods Online DNA built from modular repeats presents a challenge for gene synthesis. We present a solid surface-based sequential ligation approach, which we refer to as iterative capped assembly (ICA), that adds DNA repeat monomers individually to a growing chain while using hairpin ‘capping’ oligonucleotides to block incompletely extended chains, greatly increasing the frequency of full-length final products. Applying ICA to a model problem, construction of custom transcription activator-like effector nucleases (TALENs) for genome engineering, we demonstrate efficient synthesis of TALE DNA-binding domains up to 21 monomers long and their ligation into a nuclease-carrying backbone vector all within 3 h. We used ICA to synthesize 20 TALENs of varying DNA target site length and tested their ability to stimulate gene editing by a donor oligonucleotide in human cells. All the TALENS show activity, with the ones >15 monomers long tending to work best. Since ICA builds full-length constructs from individual monomers rather than large exhaustive libraries of pre-fabricated oligomers, it will be trivial to incorporate future modified TALE monomers with improved or expanded function or to synthesize other types of repeat-modular DNA where the diversity of possible monomers makes exhaustive oligomer libraries impractical. Oxford University Press 2012-08 2012-06-26 /pmc/articles/PMC3424587/ /pubmed/22740649 http://dx.doi.org/10.1093/nar/gks624 Text en © The Author(s) 2012. Published by Oxford University Press. http://creativecommons.org/licenses/by-nc/3.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. |
| repository_type |
Open Access Journal |
| institution_category |
Foreign Institution |
| institution |
US National Center for Biotechnology Information |
| building |
NCBI PubMed |
| collection |
Online Access |
| language |
English |
| format |
Online |
| author |
Briggs, Adrian W. Rios, Xavier Chari, Raj Yang, Luhan Zhang, Feng Mali, Prashant Church, George M. |
| spellingShingle |
Briggs, Adrian W. Rios, Xavier Chari, Raj Yang, Luhan Zhang, Feng Mali, Prashant Church, George M. Iterative capped assembly: rapid and scalable synthesis of repeat-module DNA such as TAL effectors from individual monomers |
| author_facet |
Briggs, Adrian W. Rios, Xavier Chari, Raj Yang, Luhan Zhang, Feng Mali, Prashant Church, George M. |
| author_sort |
Briggs, Adrian W. |
| title |
Iterative capped assembly: rapid and scalable synthesis of repeat-module DNA such as TAL effectors from individual monomers |
| title_short |
Iterative capped assembly: rapid and scalable synthesis of repeat-module DNA such as TAL effectors from individual monomers |
| title_full |
Iterative capped assembly: rapid and scalable synthesis of repeat-module DNA such as TAL effectors from individual monomers |
| title_fullStr |
Iterative capped assembly: rapid and scalable synthesis of repeat-module DNA such as TAL effectors from individual monomers |
| title_full_unstemmed |
Iterative capped assembly: rapid and scalable synthesis of repeat-module DNA such as TAL effectors from individual monomers |
| title_sort |
iterative capped assembly: rapid and scalable synthesis of repeat-module dna such as tal effectors from individual monomers |
| description |
DNA built from modular repeats presents a challenge for gene synthesis. We present a solid surface-based sequential ligation approach, which we refer to as iterative capped assembly (ICA), that adds DNA repeat monomers individually to a growing chain while using hairpin ‘capping’ oligonucleotides to block incompletely extended chains, greatly increasing the frequency of full-length final products. Applying ICA to a model problem, construction of custom transcription activator-like effector nucleases (TALENs) for genome engineering, we demonstrate efficient synthesis of TALE DNA-binding domains up to 21 monomers long and their ligation into a nuclease-carrying backbone vector all within 3 h. We used ICA to synthesize 20 TALENs of varying DNA target site length and tested their ability to stimulate gene editing by a donor oligonucleotide in human cells. All the TALENS show activity, with the ones >15 monomers long tending to work best. Since ICA builds full-length constructs from individual monomers rather than large exhaustive libraries of pre-fabricated oligomers, it will be trivial to incorporate future modified TALE monomers with improved or expanded function or to synthesize other types of repeat-modular DNA where the diversity of possible monomers makes exhaustive oligomer libraries impractical. |
| publisher |
Oxford University Press |
| publishDate |
2012 |
| url |
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3424587/ |
| _version_ |
1611551147618729984 |