Diagnostic probe detection system

A method for detecting the presence of a target nucleic acid sequence on a nucleic acid strand having a first target nucleic acid sequence and a second target nucleic acid sequence and distinguishing the target from an allelic variant thereof comprising the steps of:

• performing nucleic acid amplification on a sample suspected of containing said target nucleic acid sequence to produce an amplification mixture suspected of containing amplified target nucleic acid sequence;
• denaturing and neutralizing the amplification mixture suspected of containing said amplified target nucleic acid sequence;
• contacting the denatured and neutralized amplification mixture suspected of containing said amplified target nucleic acid sequence with a single probe under hybridizing conditions;
• wherein the nucleotide sequence of said single probe comprises (1) a first probe region at its 5′-end that is substantially complementary to a first target region characteristic of said target nucleic acid sequence, and (2) a second probe region, located 3′ to said first probe region, where the second probe region is substantially complementary to a second target region characteristic of said target nucleic acid sequence on the target nucleic acid strand wherein when said first target region is contiguous with said second target region on the target nucleic acid strand then the first and second probe regions on the probe are separated by a spacer region of nucleic acid, and further wherein when the first and second probe regions on the probe are contiguous then there exists an intervening sequence between the first and second target regions on the target nucleic acid strand;
• whereby for said selected hybridization conditions the first and second probe regions are such that the probe is stably hybridized to the target nucleic acid strand to form a detectable probe:
• target hybrid only when the first probe region is substantially complementary to the first target region and the second probe region is substantially complementary to the second target region,
• but wherein for said selected hybridization conditions the probe is not stably hybridized to the target nucleic acid strand to form a probe:target hybrid detectable above a threshold indicative of stable hybridization when either the first probe region is not substantially complementary to the first target region or the second probe region is not substantially complementary to the second target region;
• wherein said first and second probe regions on the probe are separated by a spacer region of nucleic acid sequence that is not complementary to the sequence of the sample nucleic acid strand between the first and second target regions on said target nucleic acid and wherein there exists an intervening sequence between the first and second target regions on said target nucleic acid that is not complementary to the sequence of the sample nucleic acid strand between the first and second probe regions on the probe;
• separating the probe from the target nucleic acid when either the first probe region is not substantially complementary to the first target region or the second probe region is not substantially complementary to the second target region;
• and
• detecting the presence or absence of the stable probe:target hybrid in the absence of elongation of the probe:target hybrid and in the absence of nicking either the probe or target as an indication of the presence of the target nucleic acid sequence in the sample,
• wherein no more than two (2) mismatches relative to the complementary sequence are present in either or both of the first and second target regions respectively.