Activating a reversibly inactivated immobilized enzyme by release from an immobilizing moiety

• Main Authors: UHLEN MATHIAS, LUNDEBERG JOAKIM
• Format: Patent
• Language: English
• Published: 26.08.2003
• Edition: 7
• Subjects: BEER; BIOCHEMISTRY; CHEMISTRY; COMPOSITIONS OR TEST PAPERS THEREFOR; COMPOSITIONS THEREOF; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL ORENZYMOLOGICAL PROCESSES; CULTURE MEDIA; ENZYMOLOGY; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIRCHEMICAL OR PHYSICAL PROPERTIES; MEASURING; MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEICACIDS OR MICROORGANISMS; METALLURGY; MICROBIOLOGY; MICROORGANISMS OR ENZYMES; MUTATION OR GENETIC ENGINEERING; ORGANIC CHEMISTRY; PEPTIDES; PHYSICS; PROCESSES OF PREPARING SUCH COMPOSITIONS; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; SPIRITS; TECHNICAL SUBJECTS COVERED BY FORMER USPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ARTCOLLECTIONS [XRACs] AND DIGESTS; TESTING; VINEGAR; WINE

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.