Analysis of interactions of template/primer duplexes with T7 DNA polymerase by oligonucleotide microarray

Abstract

Recent advances in DNA microarray technology have contributed significantly to assessing massive quantities of genetic data, especially for gene expression monitoring, mutation detection and polymorphism analysis. However, fewer studies have been reported on the use of such novel technique for molecular recognition events, involving the protein–DNA interactions. In this study, DNA microarray technology was employed to study the binding interactions between a series of template/primer DNA duplexes (dsDNA) and single-stranded DNA (ssDNA) and T7 DNA polymerase enzymes containing both polymerase and exonuclease activities. Various DNA targets were printed on the streptavidin-modified gold surface based on the biotin–streptavidin chemistry. The effects of mismatches on the overall binding affinities of T7 DNA polymerase were estimated after introduction of a number of different mismatches at/near the 3′-end of the primer strand. The overall binding affinities of the enzyme towards the mismatched DNAs were one to four times less than with the fully matched DNA. The results have demonstrated the feasibility of using the microarray technique to study molecular recognition events such as the mismatch base discrimination involved in DNA–polymerase interactions.