Characterization and engineering of a DNA polymerase reveals a single amino-acid substitution in the fingers subdomain to increase strand-displacement activity of A-family prokaryotic DNA polymerases

摘要

BackgroundThe discovery of thermostable DNA polymerases such as Taq DNA polymerase revolutionized amplification of DNA by polymerase chain reaction methods that rely on thermal cycling for strand separation. These methods are widely used in the laboratory for medical research, clinical diagnostics, criminal forensics and general molecular biology research. Today there is a growing demand for on-site molecular diagnostics; so-called ‘Point-of-Care tests’. Isothermal nucleic acid amplification techniques do not require a thermal cycler making these techniques more suitable for performing Point-of-Care tests at ambient temperatures compared to traditional polymerase chain reaction methods. Strand-displacement activity is essential for such isothermal nucleic acid amplification; however, the selection of DNA polymerases with inherent strand-displacement activity that are capable of performing DNA synthesis at ambient temperatures is currently limited.