Patent ID: 6403313
Filing Date: 2002-06-11
Classification: C12Q

Abstract:
A method for assaying binding, said method comprising:providing a target comprising at least one nucleic acid sequence; providing a probe comprising a nucleic acid or nucleic acid analog sequence incompletely complementary to at least a portion of said target; providing an intercalating agent, wherein either said probe or said intercalating agent comprises a fluorophore; adding said probe, said target and said intercalating agent to a hybridization medium to provide a test sample comprising triplexes of said probe and said target, wherein said triplexes comprise base triplets, and all said base triplets of said triplexes are members selected from the group consisting of A-T-A, T-A-T, U-A-T, T-A-U, A-U-A, U-A-U, G-C-G and C-G-C, and wherein at least one of said base triplets is T-A-T, U-A-T, T-A-U, U-A-U or C-G-C, and at least one other of said base triplets is A-T-A, G-C-G or A-U-A; irradiating said test sample with exciting radiation to cause said fluorophore to emit fluorescent radiation; detecting an intensity of said fluorescent radiation, wherein said intensity increases with increasing binding affinity between said probe and said target; and determining from said intensity a number of mismatched bases, an identity of a mismatched base and/or a location of a mismatch between said probe and said target to assay binding, wherein said determining is accomplished by calibrating said intensity against intensities exhibited by reference probes combined with said target and said intercalating agent, said reference probes differing from each other by at least one base, wherein said method is conducted without denaturing said target or said probe.