METHOD FOR ACTIVATING A REVERSIBLY INACTIVATED IMMOBILIZED ENZYME BY RELEASE FROM AN IMMOBILISING MOIETY AND ITS USE IN NUCLEICACID AMPLIFICATION REACTIONS

摘要

Enzymes or catalytic fragments thereof reversibly inactivated by attachment to an immobilizing moiety which may comprise a magnetic particle are activated by release from the immobilizing moiety. The enzyme or fragment may be in the form of a fusion protein that is attached to the immobilizing moiety via a pair of affinity binding partners such that the enzyme or fragment is reversibly inactivated, and release of the fusion protein from the immobilizing moiety activates the enzyme or fragment. Enzymes include DNA polymerases, DNA ligases, Reverse transcriptases and RNA polymerases. The enzyme or fragment may be thermostable, and the fusion protein can be bound to the immobilizing moiety via a heat-labile linkage. Activation of the reversibly inactivated immobilized enzyme or fragment has particular utility in PCR and analogous nucleic acid amplification techniques. A sample containing a nucleic acid is contacted with the immobilized fusion protein, and release of the fusion protein activates the enzyme or fragment to start a first cycle of an amplification reaction. Amplification reactions that may be carried out include Ligase chain reaction (LCR), Self-sustained Sequence Replication (3SR), Reverse transcriptase PCR (RT-PCR), Q-beta replicase amplification reaction and nucleic acid sequence-based amplification (NASBA). Kits for the amplifications may be prepared containing an appropriate reversibly immobilized enzyme in the form of a fusion protein, and primers and/or probe for performing the amplification.