Journal: Nucleic Acids Research

Article Title: Removal of mismatched bases from synthetic genes by enzymatic mismatch cleavage

doi: 10.1093/nar/gni058

Figure Lengend Snippet: Synthesis of a functional chloramphenicol acetyltransferase gene with changed codon composition. The ratio r of ‘active clones’ to ‘analyzed clones’ as described in the text is shown for different gene synthesis methods with or without an EMC step. A significant increase of r can be observed only in the cases where EMC is combined with an exonuclease activity present in the reaction or in the later amplification reaction. Prolonged incubation with E.coli endonuclease V results in no detectable product after the amplification steps (ss, single-stranded synthesis, ds, double-stranded synthesis; VII, T4 endonuclease VII; V, E.coli endonuclease V; T, Taq DNA polymerase; and Vn, Vent DNA polymerase).

Article Snippet: Synthesis of double-stranded DNA was performed by thermal cycling of 10 μl phosphorylated sense oligonucleotides, 10 μl phosphorylated antisense oligonucleotides, 3 μl 10× Taq DNA ligase buffer (NEB) in a total volume of 30 μl.

Techniques: Functional Assay, Clone Assay, Activity Assay, Amplification, Incubation