T7 Endonuclease I Mediates Error Correction in Artificial Gene Synthesis

Area of Science:

• Molecular Biology
• Synthetic Biology

Background:

• De novo gene synthesis relies on high-quality oligonucleotides for PCR assembly.
• Current gene synthesis protocols have limitations in error rates, impacting efficiency and accuracy.

Purpose of the Study:

• To evaluate the efficacy of endonuclease enzymes in removing mutations from synthetic genes.
• To improve the fidelity of de novo gene synthesis through an enzymatic mismatch cleavage (EMC) step.

Main Methods:

• Artificial synthesis of the green fluorescent protein (gfp) gene.
• Integration of an enzymatic mismatch cleavage (EMC) step using T7 endonuclease I post-gene assembly.
• Functional and sequence analysis of synthesized genes.

Main Results:

• T7 endonuclease I significantly reduced deletions, insertions, and substitutions in synthetic genes.
• Mutation frequency was reduced eightfold compared to protocols without error correction.
• Enzymatic mismatch cleavage achieved an error frequency of 0.43 errors per kilobase.

Conclusions:

• Enzymatic mismatch cleavage with T7 endonuclease I effectively enhances the fidelity of artificial gene synthesis.
• This approach improves the yield of error-free synthetic genes.
• Incorporating mutation removal steps is crucial for accurate de novo gene synthesis.