Comprehensive Profiling of Four Base Overhang Ligation Fidelity by T4 DNA Ligase and Application to DNA Assembly

ACS Synth Biol. 2018 Nov 16;7(11):2665-2674. doi: 10.1021/acssynbio.8b00333. Epub 2018 Oct 29.


Synthetic biology relies on the manufacture of large and complex DNA constructs from libraries of genetic parts. Golden Gate and other Type IIS restriction enzyme-dependent DNA assembly methods enable rapid construction of genes and operons through one-pot, multifragment assembly, with the ordering of parts determined by the ligation of Watson-Crick base-paired overhangs. However, ligation of mismatched overhangs leads to erroneous assembly, and low-efficiency Watson Crick pairings can lead to truncated assemblies. Using sets of empirically vetted, high-accuracy junction pairs avoids this issue but limits the number of parts that can be joined in a single reaction. Here, we report the use of comprehensive end-joining ligation fidelity and bias data to predict high accuracy junction sets for Golden Gate assembly. The ligation profile accurately predicted junction fidelity in ten-fragment Golden Gate assembly reactions and enabled accurate and efficient assembly of a lac cassette from up to 24-fragments in a single reaction.

Keywords: DNA assembly; DNA ligase fidelity; DNA ligases; Golden Gate assembly; single-molecule sequencing.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Pairing
  • DNA / chemistry
  • DNA / metabolism*
  • DNA Ligases / metabolism
  • Lac Operon / genetics
  • Synthetic Biology / methods*


  • DNA
  • DNA Ligases