UNIT 3.23 Design and Assembly of Large Synthetic DNA Constructs

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

Published Online: 1 JUL 2012

DOI: 10.1002/0471142727.mb0323s99

Current Protocols in Molecular Biology

Current Protocols in Molecular Biology

How to Cite

Miklos, A. E., Hughes, R. A. and Ellington, A. D. 2012. Design and Assembly of Large Synthetic DNA Constructs. Current Protocols in Molecular Biology. 99:V:3.23:3.23.1–3.23.18.

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


The availability of custom synthetic gene-length DNA products removes numerous bottlenecks in research efforts, making gene synthesis an increasingly common commercial service. However, the assembly of synthetic oligonucleotides into large, custom DNA constructs is not especially difficult, and performing “in-house” gene synthesis has time and cost advantages. This unit will treat both the concerns of design and physical assembly in gene synthesis, including how to design DNA sequences for synthesis and the design of overlapping oligonucleotide schemes to ensure facile assembly into the final product. Assembly is accomplished using a reliable series of PCR reactions, with a troubleshooting assembly protocol included, which not only assembles difficult sequences but allows identification of the source of a failure down to a pair of oligonucleotides. Curr. Protoc. Mol. Biol. 99:3.23.1-3.23.18. © 2012 by John Wiley & Sons, Inc.


  • gene synthesis;
  • synthetic genes;
  • synthetic DNA;
  • synthetic biology;
  • protein expression;
  • gene assembly;
  • oligonucleotides;
  • oligonucleotide assembly;
  • oligonucleotide synthesis;
  • inside-out nucleation;
  • PCR