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

Citations Over TimeTop 10% of 2018 papers

Abstract

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.

Related Papers

• → Sealing of gaps in duplex DNA by T4 DNA ligase(1982)74 cited
• → Effects of base mismatches on joining of short oligodeoxynucleotides by DNA ligases(1997)61 cited
• → Specific and reversible inhibition of the blunt end joining activity of the T4 DNA ligase(1981)30 cited
• → DNA Ligases(1989)2 cited
• → DNA Ligation and Escherichia coli Transformation(1986)