Development and Application of Antibody Modified DNA Sequencing Enzymes

doi:10.13560/j.cnki.biotech.bull.1985.2023-1103

Abstract

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, sequencing

SONG Hui, CAO Wen-gang, XIAO Xiao-wen, DU Jun. Development and Application of Antibody Modified DNA Sequencing Enzymes[J]. Biotechnology Bulletin, 2024, 40(5): 84-93.

Figures/Tables 6

Fig. 1 Schematic diagram of gene structure of DNA sequencing enzyme Sso-Sequenase

Fig. 2 Purification of Sso-Sequenase protein

Fig. 3 STR detection of Sso-Sequenase

Fig. 4 Performance of DNA sequencing enzyme Sso-Sequenase on repeat sequence templates

Fig. 5 Performance of DNA sequencing enzyme Sso-Sequenase on high GC templates

Fig. 6 Performance of DNA sequencing enzyme Sso-Sequenase on hairpin structure templates

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