Detection of mismatches by resolvase cleavage using a magnetic bead

Abstract

Disclosed is a method for detecting one or more mismatches between a first and a second nucleic acid, the first and second nucleic acids being capable of preferentially hybridizing. The method involves: a) providing the first nucleic acid in its single-stranded form, the first nucleic acid being bound to the first member of a specific binding pair; b) providing the second nucleic acid in its single-stranded form, the second nucleic acid being bound to a detectably labelled reagent; c) contacting the first nucleic acid with the second nucleic acid under conditions allowing heteroduplex formation; d) contacting the product of step (c) with a magnetic bead to which is bound the second member of the specific binding pair under conditions allowing complex formation between the first and the second members of the specific binding pair; e) applying a magnetic field to the mixture to facilitate separation of the magnetic bead from the remainder of the product of step (c); f) contacting the magnetic bead-bound nucleic acid with a resolvase capable of recognizing at least one single base pair mismatch in a heteroduplex, under conditions which permit the resolvase to cleave the heteroduplex; and f) analyzing the product of step (f), the presence of a cleavage product being an indication of a mismatch between the first and second nucleic acids.