In situ overlap and sequence synthesis during DNA assembly

ACS Synth Biol. 2013 Dec 20;2(12):750-5. doi: 10.1021/sb400067v. Epub 2013 Nov 6.


Modern cloning methods are independent from restriction enzyme recognition sites. However, nearly all current cloning methods still require the introduction of overlaps by PCR, which can introduce undesired mutations. Here, we investigated whether overlaps needed for DNA assembly can be synthesized in situ and we tested if the de novo synthesis of sequences can be simultaneously combined with the assembly of larger double-stranded DNA fragments. We showed in a set of 44 cloning experiments that overlaps of 20 bp needed for DNA assembly can be synthesized in situ from single-stranded oligonucleotides. Short sequences of 30-255 bp can be synthesized from single-stranded oligonucleotides concurrently with DNA assembly, and both techniques can be combined. The assembly of similar constructs by state-of-the-art techniques would have required multiple rounds of cloning or tedious sample preparations, whereas our approach is a one-step reaction.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cloning, Molecular / methods*
  • DNA* / chemistry
  • DNA* / metabolism
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Genetic Vectors / genetics
  • Oligonucleotides* / chemistry
  • Oligonucleotides* / metabolism


  • Oligonucleotides
  • DNA