Abstract: 【Objective】To develop a novel DNA sequencing enzyme that addresses the main challenges faced by first-generation sequencing technology when dealing with complex DNA structure templates, such as interruption of sequencing signals or rapid signal decay.【Method】Taq DNA polymerase and single-strand binding protein SSB gene sequences were mined from the NCBI database, and a novel sequencing enzyme, Sso-Sequenase, was obtained using genetic fusion, site-directed mutagenesis, and gene design techniques. The purified sequencing enzyme was obtained through affinity chromatography and ion exchange chromatography. The performance of Sso-Sequenase was improved using antibody modification technology, and its hot start performance was characterized using STR technology. A variety of complex templates were selected, and the sequencing performance of the Sso-Sequenase sequencing enzyme kit was compared with the traditional BigDye sequencing kit using Sanger sequencing.【Result】Sso-Sequenase was stably expressed in Escherichia coli, with a purity of over 95% and a yield of up to 10.5 mg/L. When the temperature was below 35℃ , Sso-Sequenase demonstrated hot start activity. In first-generation sequencing reactions with complex templates, such as those with repetitive sequences, high GC content, and hairpin structures, Sso-Sequenase successfully completed sequencing for all samples, with an average base quality value QV > 20. In contrast, the BigDye sequencing kit experienced significant signal decay or interruption when processing these complex samples.【Conclusion】A DNA sequencing enzyme, Sso-Sequenase, has been developed to possess exceptional purity, remarkable yield, and hot-start activity. These advancements have significantly bolstered the sequencing success rate for intricate DNA templates, including those characterized by repetitive sequences, high GC content, and hairpin structures.

Key words: DNA sequencing enzyme, antibody modification, complex templates, first-generation sequencing