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US 8,512,953 B2Granted
Diagnostic probe detection system
AssigneeOne Lambda
InventorsKatsuyuki Saito, Jar-How Lee, Lindley Blair
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Abstract
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 said 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.
Claims18
A method for detecting the presence of two or more target nucleic acid sequences on two or more sample nucleic acid strands comprising the steps of:
- contacting a sample suspected of containing said target nucleic acid sequences with a diagnostic probe under hybridizing conditions;
- wherein the first target nucleic acid sequence has a first target region and a first complementary target zone;
- wherein the second nucleic acid sequence has a second target region and a second complementary target zone;
- wherein the nucleotide sequence of said diagnostic probe comprises (1) a first probe region that is substantially complementary to a first target region characteristic of said first target nucleic acid sequence, and (2) a second probe region, where the second probe region is substantially complementary to a second target region characteristic of said second target nucleic acid sequence;
- wherein said first and second probe regions on the diagnostic probe may be separated by a spacer region of nucleic acid wherein when said first and second probe regions are separated by a spacer region then said spacer region forms a non-self-hybridized loop under said selected conditions;
- whereby for said selected hybridization conditions the first and second probe regions are such that the diagnostic probe is only stably hybridized to the target nucleic acid strand to form a detectable probe:target hybrid when the first complementary target zone is substantially complementary to the second complementary target zone, 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 diagnostic 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 complementary target zone is not substantially complementary to the second complementary target zone, or 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;
- separating the diagnostic probe from the target nucleic acid when either the first probe region is not exactly complementary to the first target region or the second probe region is not exactly 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 as an indication of the presence of the target nucleic acid sequences in the sample.
7 more elements
A method for detecting the presence of two or more target nucleic acid sequences on two or more sample nucleic acid strands comprising the steps of:
- contacting a sample suspected of containing said target nucleic acid sequences with a diagnostic probe under hybridizing conditions;
- wherein the first target nucleic acid sequence has a first target region and a first complementary target zone;
- wherein the second nucleic acid sequence has a second target region and a second complementary target zone;
- wherein the nucleotide sequence of said diagnostic probe comprises (1) a first probe region that is substantially complementary to a first target region characteristic of said first target nucleic acid sequence, and (2) a second probe region, where the second probe region is substantially complementary to a second target region characteristic of said second target nucleic acid sequence;
- wherein said first and second probe regions on the diagnostic probe may be separated by a spacer region of nucleic acid wherein when said first and second probe regions are separated by a spacer region then said spacer region forms a non-self-hybridized loop under said selected conditions;
- whereby for said selected hybridization conditions the first and second probe regions are such that the diagnostic probe is only stably hybridized to the target nucleic acid strand to form a detectable probe:target hybrid when the first complementary target zone is substantially complementary to the second complementary target zone, 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 diagnostic 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 complementary target zone is not substantially complementary to the second complementary target zone, or 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;
- separating the diagnostic probe from the target nucleic acid when either the first probe region is not exactly complementary to the first target region or the second probe region is not exactly 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 sequences in the sample.
7 more elements
Dependencies are inferred from the claim text.