Enrichment of Error‐Free Synthetic DNA Sequences by CEL I Nuclease

Randall A. Hughes1, Aleksandr E. Miklos1, Andrew D. Ellington1

1 The University of Texas at Austin, Applied Research Laboratories, Department of Chemistry and Biochemistry, Center for Systems and Synthetic Biology, Austin, Texas
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 3.24
DOI:  10.1002/0471142727.mb0324s99
Online Posting Date:  July, 2012
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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

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Cel I Endonuclease Treatment of Synthetic DNA
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Cel I Endonuclease Treatment of Synthetic DNA

  Materials
  • Designed gene (up to 2 kbp, or a DNA fragment of equivalent size)
  • PCR clean‐up (purification) kit (Promega Wizard SV Gel and PCR Cleanup kit, Promega, cat. no. A9282, QiaQuick PCR Purification kit, Qiagen, cat. no. 28106, or similar)
  • 10× Taq polymerase buffer (NEB; 1×: 10 mM Tris⋅Cl, pH 8.3, 50 mM KCl, 1.5 mM MgCl 2)
  • Molecular biology‐grade (nuclease‐free) water
  • Ice
  • SURVEYOR Mutation Detection kit (Transgenomic, cat. no. 706025) containing:
    • Enhancer S solution (Surveyor Enhancer S)
    • CEL I nuclease (Surveyor Nuclease S)
    • Stop Solution (Surveyor Stop Solution)
  • Low‐melting temperature agarose
  • 6× DNA loading dye (see recipe in unit 2.5)
  • dsDNA ladder of choice (with appropriate size range)
  • 5× Phusion HF Buffer (New England Biolabs)
  • dNTP (deoxynucleotide triphosphate) mixture (4 mM of each)
  • 25 µM stock of Forward (5′) flanking primer to DNA of interest
  • 25 µM stock of Reverse (3′) flanking primer to DNA of interest
  • Phusion Hi‐Fidelity DNA Polymerase (New England Biolabs)
  • Analytical‐grade agarose
  • 200‐µl thin‐walled nuclease‐free PCR tubes
  • Thermal cycler
  • Microcentrifuge
  • Heat block, optional
  • 1.5‐ or 2‐ml microcentrifuge tubes
  • Clean razor blade
  • Additional reagents and equipment for assembling the designed gene using a method of choice (Chapter 3), quantifying the purified DNA ( appendix 3D or appendix 3J), and agarose gel electrophoresis (unit 2.5)
NOTE: Primers can be of any‐length as long as they have been designed to be compatible with each other in PCR (low secondary structure, little hetero‐ and homo‐dimer formation propensity and annealing temps within 5°C of one another). Generally, primers 20 to 25 bp with annealing temperatures 60° to 65°C work well.
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Figures

Videos

Literature Cited

Literature Cited
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