Unit

UNIT 3.24 Enrichment of Error-Free Synthetic DNA Sequences by CEL I Nuclease

  1. Randall A. Hughes,
  2. Aleksandr E. Miklos,
  3. Andrew D. Ellington

Published Online: 1 JUL 2012

DOI: 10.1002/0471142727.mb0324s99

Current Protocols in Molecular Biology

Current Protocols in Molecular Biology

How to Cite

Hughes, R. A., Miklos, A. E. and Ellington, A. D. 2012. Enrichment of Error-Free Synthetic DNA Sequences by CEL I Nuclease. Current Protocols in Molecular Biology. 99:V:3.24:3.24.1–3.24.10.

Author Information

  1. The University of Texas at Austin, Applied Research Laboratories, Department of Chemistry and Biochemistry, Center for Systems and Synthetic Biology, Austin, Texas

Publication History

  1. Published Online: 1 JUL 2012
  2. Published Print: JUL 2012

Abstract

As the availability of DNA sequence information has grown, so has the need to replicate DNA sequences synthetically. Synthetically produced DNA sequences allow the researcher to exert greater control over model systems and allow for the combinatorial design and construction of novel metabolic and regulatory pathways, as well as optimized protein-coding sequences for biotechnological applications. This utility has made synthetically produced DNA a hallmark of the molecular biosciences and a mainstay of synthetic biology. However, synthetically produced DNA has a significant shortcoming in that it typically has an error rate that is orders of magnitude higher when compared to DNA sequences derived directly from a biological source. This relatively high error rate adds to the cost and labor necessary to obtain sequence-verified clones from synthetically produced DNA sequences. This unit describes a protocol to enrich error-free sequences from a population of error-rich DNA via treatment with CEL I (Surveyor) endonuclease. This method is a straightforward and quick way of reducing the error content of synthetic DNA pools and reliably reduces the error rates by >6-fold per round of treatment. Curr. Protoc. Mol. Biol. 99:3.24.1-3.24.10. © 2012 by John Wiley & Sons, Inc.

Keywords:

  • CEL I;
  • error correction;
  • synthetic DNA;
  • Surveyor nuclease;
  • synthetic biology;
  • oligonucleotide;
  • oligonucleotide synthesis