Plasmid DNA serves as the most commonly used gene delivery vehicle,playing a crucial role in gene synthesis tech-nology.Achieving accurate and rapid detection of synthesized plasmid DNA is the key to ensuring the integrity of the genome and improving the efficiency of gene synthesis.DNA detection methods based on first-generation sequencing have established their ac-curacy as an industry standard,but they have limitations in terms of detection throughput,speed,and cost,which has prompted scientists to continuously seek new solutions.Based on the biological enzyme library,the DNA library construction enzyme TN5 was developed,and a high-throughput plasmid DNA detection solution,named Fast NGS,was established.The feasibility of Fast NGS was evaluated using plasmid DNA samples of different lengths and qualities and high-throughput sequencing of plasmid DNA samples was performed.Finally,the efficiency of Fast NGS and Sanger sequencing was compared.The results showed that the purity and quality of the DNA library construction enzyme TN5 protein met the requirements of next-generation sequencing.Fast NGS is suitable for sequencing of 3～8 kb gene synthesis plasmids,and its detection throughput is up to 2 500 per 12 h,with a sequencing success rate of over 95%.The sequencing accuracy is comparable to that of first-generation sequencing,and there is no significant sequence preference.Fast NGS achieves high-throughput,rapid,and low-cost detection of plasmid DNA,pro-viding a new direction for the development of gene synthesis technology.
关键词
Fast/NGS/质粒DNA/高通量测序/TN5
Key words
Fast NGS/plasmid DNA/high-throughput sequencing/TN5
维普
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