Thermally reversible inactivation of Taq polymerase in an organic solvent for application in hot start PCR

Abstract

In the past, Taq polymerase was reversibly inactivated by modification with a dicarboxylic acid anhydride in aqueous media, to enable ‘hot start PCR’. However, there are various constraints in using such a method including temperature and concentration. Here we describe an alternative method whereby Taq polymerase may be reversibly inactivated following incubation with an excess of citraconic anhydride at elevated temperatures, in an anhydrous non-protic organic solvent – tert-butyl methyl ether – by first drying the enzyme with a salt or carbohydrate excipient to form an amorphous powder. Reactivation of the enzyme is due to the instability of the chemical modification at low pH following a short incubation in a suitable buffer.