T7 Endonuclease I Mediates Error Correction in Artificial Gene Synthesis

Abstract

Efficacy of de novo gene synthesis largely depends on the quality of overlapping oligonucleotides used as template for PCR assembly. The error rate associated with current gene synthesis protocols limits the efficient and accurate production of synthetic genes, both in the small and large scales. Here, we analysed the ability of different endonuclease enzymes, which specifically recognize and cleave DNA mismatches resulting from incorrect impairments between DNA strands, to remove mutations accumulated in synthetic genes. The gfp gene, which encodes the green fluorescent protein, was artificially synthesized using an integrated protocol including an enzymatic mismatch cleavage step (EMC) following gene assembly. Functional and sequence analysis of resulting artificial genes revealed that number of deletions, insertions and substitutions was strongly reduced when T7 endonuclease I was used for mutation removal. This method diminished mutation frequency by eightfold relative to gene synthesis not incorporating an error correction step. Overall, EMC using T7 endonuclease I improved the population of error-free synthetic genes, resulting in an error frequency of 0.43 errors per 1 kb. Taken together, data presented here reveal that incorporation of a mutation-removal step including T7 endonuclease I can effectively improve the fidelity of artificial gene synthesis.

A systematic comparison of error correction enzymes by next-generation sequencing

“…We find that the addition of T7 ligase actually decreased assembly quality relative to the no ligase control. In agreement with previous studies, we also find T7 Endonuclase I to be highly sensitive to protocol and concentration as exhibited by the wide range of error frequencies [12,14]. After T7 Endonuclease I, we found MutS to be the third most effective enzyme at 11.2 errors/kb, with T4 Endonuclease VII, Surveyor, and Endonuclease V following.…”

“…With our method tuned, we then analyzed errors in model a two-oligo assembly to assess its sensitivity. Previous studies found the most common errors in the final gene synthesis product to be either single-base deletions [11,14,19], or mismatches [12,20]. Here, we also found mismatches to be the most common error in our assembly.…”

“…Previous assessments of different enzymatic error correction methods have relied on Sanger sequencing of finished gene synthesis products to determine their efficiencies [11,12,14,19,20].…”

“…Although these oligos are of high enough quality for common applications such as PCR, their error rates make practical gene synthesis challenging. Several groups have managed to synthesize genes from such oligos, but only find about 5-60% perfect products depending on the size and complexity of the template [11,12,13,14]. This problem is further exacerbated when using lower-cost, but often lower quality oligos from array-based synthesis approaches [15,16,17,18,19,20].Consequently, researchers have developed a number of methods to ameliorate oligo error rate post-synthesis.…”

Stability and Ligand Promiscuity of Type A Carbohydrate-binding Modules Are Illustrated by the Structure of Spirochaeta thermophila StCBM64C

“…Gene synthesis was used to produce 56 genes for which there was no access to a gDNA template. Artificial genes were designed with a codon usage table optimized for expression in E. coli and synthesized using standard procedures (23). Purified nucleic acids (ϳ50 ng) were subsequently cloned into pHTP9 E. coli expression vector using the NZYEasy cloning kit (NZYTech Ltd.), according to established protocols for the ligation independent cloning technology.…”