Helicase Purification for DNA Sequencing

Abstract

BACKGROUND: A method to increase accuracy and ease-of-use, while decreasing time and cost in deoxyribonucleic acid (DNA) sequence identification, is sought after. Helicase, which unwinds DNA, and avidin, which strongly attracts biotin for potential attraction of biotinylated DNA segments, were investigated for use in a novel DNA sequencing method. AIM: This study aimed to (1) purify bacteriophage T7 gene product 4 helicase and helicase-avidin fusion protein in a bacterial host and (2) characterize their functionality. METHODS: Helicase and helicase-avidin were cloned for purification from bacteria. Helicase-avidin was solubilised via urea denaturation/renaturation. DNA and biotin binding were assessed using Electrophoretic Mobility Shift Assays and biotinylated resins, respectively. RESULTS: (1) Helicase and helicase-avidin proteins were successfully purified. (2) Helicase protein was able to bind DNA and avidin protein strongly bound biotin. CONCLUSION: Helicase and helicase-avidin can be purified in a functional form from a bacterial host, thus supporting further investigation for DNA sequencing purposes.