Optimization of PCA Error Correction Conditions to Improve Efficiency of Virus Genome De Novo Synthesis

In recent years, there have been frequent global outbreaks of viral epidemics such as Zika, COVID-19, and monkeypox, which have had a huge impact on human health and society and have also spurred innovation in virus engineering technology. The rise of synthetic virus genome technology has provided researchers with a new platform to accelerate vaccine and drug development. Although DNA synthesis technology has made significant progress, the current virus genome synthesis technology still requires the assembly of short oligonucleotides of around 60 bp into kb-level lengths when constructing long segments, a process in which the commonly used polymerase chain reaction assembly (PCA) technology has high error rates and is cumbersome to operate. This study optimized the error correction conditions after PCA assembly, increasing the accuracy of synthesizing 1 kb DNA fragments from 4.2 ± 2.1% before error correction to 31.3 ± 3.1% after two rounds of correction, an improvement of over 6 times. This study provides a more efficient operational process for synthesizing virus genomes from scratch, indicating greater potential for virus engineering in epidemic prevention and control and the field of biomedicine.