Diagnostic probe detection system
The invention provides methods for detecting target nucleic acid sequences with diagnostic probes including first and second probe regions that are substantially complementary to first and second target regions respectively on a target nucleic acid strand wherein the first probe region is located 5′ to the second probe region. The first probe region is substantially complementary to the first target region, on the target nucleic acid strand, which also includes a second target region, wherein when the first target region is contiguous with the second target region on the target nucleic acid strand, then the first and second probe regions on the diagnostic probe are separated by a spacer region of nucleic acid.
1. 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.
2. The method of claim 1 wherein said spacer is from 1 to 30 bases long.
3. The method of claim 1 wherein said spacer is from 3 to 10 bases long.
4. The method of claim 1 wherein said first target region is from 1 to 350 bases from said second target region on the target nucleic acid sequence.
5. The method of claim 1 wherein the target nucleic acid sequence is characteristic of one or more human leukocyte antigens (HLA) or T-cell receptor (TCR) gene sequences.
6. The method of claim 1 wherein the sample suspected of containing said target nucleic acid is derived from a human being.
7. The method of claim 1 wherein the first and second probe regions, or first or second probe regions, each comprise no more than one (1) mismatch relative to the complementary sequence in the first and second target regions respectively.
8. 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:
contacting a denatured and neutralized nucleic acid amplification mixture suspected of containing 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 each of the first and second target regions respectively.
9. The method of claim 8 wherein said spacer is from 1 to 30 bases long.
10. The method of claim 8 wherein said spacer is from 3 to 10 bases long.
11. The method of claim 8 wherein said first target region is from 1 to 350 bases from said second target region on the target nucleic acid sequence.
12. The method of claim 8 wherein the target nucleic acid sequence is characteristic of one or more human leukocyte antigens (HLA) or T-cell receptor (TCR) gene sequences.
13. The method of claim 8 wherein the sample suspected of containing said target nucleic acid is derived from a human being.
14. The method of claim 8 wherein the first and second probe regions, or first or second probe regions, each comprise no more than one (1) mismatch relative to the complementary sequence in the first and second target regions respectively.