Reversibly modified thermostable enzymes for DNA synthesis and amplification in vitro

US7378262B2 · kind B2 · utility

Assignee

• Roche Molecular Systems, Inc. · US

Inventors

• Harald Sobek · Penzberg, DE
• Michael Greif · Penzberg, DE

Key dates

• Filing date: Dec 11, 2002
• Grant date: May 27, 2008
• Expiry date: Nov 27, 2023

Classification

• Technology area (CPC C): Chemistry; Metallurgy
• CPC primary: C12Q1/686
• WIPO field: Biotechnology
• WIPO sector: Chemistry

Abstract

The invention relates to a composition comprising a first modified thermostable enzyme exhibiting 3′exonuclease activity but essentially no DNA polymerase activity and a second modified thermostable enzyme exhibiting DNA polymerase activity, whereas the fidelity of an amplification process is enhanced by the use of the composition in an amplification process in comparison to the use of the single second enzyme in an amplification process and, whereas said first and said second modified thermostable enzyme is reversibly modified by an inhibiting agent which results in essentially complete inactivation of enzyme activity, wherein incubation of said first and said second modified thermostable enzyme in an aqueous buffer at alkaline pH at a temperature less than 25° C. for 20 minutes results in no significant increase in the activity of said first and said second modified thermostable enzyme, wherein incubation at a temperature greater than 50° C. in an aqueous buffer at alkaline pH results in at least tow-fold increase in enzyme activity in less than 20 minutes which allow formation of primer extension products.