High-fidelity PCR enzyme with DNA-binding domain facilitates de novo gene synthesis

Nowadays enzymatic synthesis of genes is the most powerful tool for fast resolution of the various tasks in the field of basic and applied biological research. PCR-based gene assembly from overlapping oligonucleotides has become a widely used strategy. However, all the methods described in the literature are not perfect and need an extra processing step. In this study we are verifying Phusion high-fidelity polymerase as a tool to reduce nucleotide mismatches in de novo gene synthesis, thus facilitating subsequent cloning. To test the efficiency of the polymerase, we selected Fel d 4 gene, which is a 581 bp DNA sequence encoding the lipocalin allergen protein, one of the major cat allergens. The approach described here, therefore, would be useful in DNA sequences creation.