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Nucleic Acids Res DOI:10.1093/nar/gks624

Iterative capped assembly: rapid and scalable synthesis of repeat-module DNA such as TAL effectors from individual monomers.

Publication TypeJournal Article
Year of Publication2012
AuthorsBriggs, AW, Rios, X, Chari, R, Yang, L, Zhang, F, Mali, P, Church, GM
JournalNucleic Acids Res
Volume40
Issue15
Pagese117
Date Published2012 Aug
ISSN1362-4962
KeywordsBase Sequence, Cell Line, Deoxyribonucleases, Type II Site-Specific, DNA, DNA-Binding Proteins, Genes, Reporter, Genes, Synthetic, Homologous Recombination, Humans, Molecular Sequence Data, Protein Structure, Tertiary, Repetitive Sequences, Nucleic Acid
Abstract

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.

URLhttp://nar.oxfordjournals.org/cgi/pmidlookup?view=long&pmid=22740649
DOI10.1093/nar/gks624
Pubmed

http://www.ncbi.nlm.nih.gov/pubmed/22740649?dopt=Abstract

Alternate JournalNucleic Acids Res.
PubMed ID22740649
PubMed Central IDPMC3424587
Grant ListT32 GM007753 / GM / NIGMS NIH HHS / United States
1P50 HG005550 / HG / NHGRI NIH HHS / United States