抗体修饰DNA测序酶的开发及其应用OA北大核心CSTPCD

[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.