Methods and compositions for universal detection of nucleic acids
First Claim
1. A method of detecting the presence or amount of a first target nucleic acid in a sample, the method comprising:
- (a) linking first and second universal DNA segments into a first molecule in a linking reaction dependent on the first target nucleic acid, thereby providing a first tagged target nucleic acid; and
(b) PCR amplifying the first tagged target nucleic acid using first and second universal primers, each universal primer having a 3′
portion that anneals to one of the two universal DNA segments;
wherein;
the 3′
portion of the first universal primer anneals to the first universal segment;
a 5′
portion of the first universal primer comprises a nucleic acid sequence sufficiently similar to a portion of the first universal DNA segment, a portion of the second universal DNA segment, or a portion of the first target nucleic acid, to anneal to a complement of the portion of the first universal DNA segment, a complement of the portion of the second universal DNA segment, or a complement of the portion of the first target nucleic acid, respectively, under PCR reaction conditions;
an amplicon generated upon extension of the first universal primer forms an intramolecular hairpin stem between the 5′
portion of the first universal primer and a portion of the amplicon complementary to the first universal DNA segment, the second universal DNA segment, or the first target nucleic acid;
or wherein the first universal primer forms a circular structure upon annealing to the first tagged nucleic acid that brings the 3′ and
5′
-ends of the first universal primer close to each other; and
,formation of the hairpin stem or circular structure results or causes a change in a first detectable signal, the first detectable signal indicating the presence or quantity of the first target nucleic acid in the sample.
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Accused Products
Abstract
Provided are methods and compositions for detecting the presence or amount of one or more target nucleic acids in a sample. Methods of the present invention include linking universal nucleic acid segments into a single molecule in a linking reaction dependent on a target nucleic acid of interest. A variety of universal segment linking strategies are provided, including preamplification by polymerase chain reaction, ligation-based strategies, reverse transcription and linear polymerase extension. Linking the universal segments into a single molecule generates a tagged target nucleic acid which is detected in a manner dependent on an intramolecular interaction between one universal segment and a second portion of the tagged target nucleic acid. In certain embodiments, the intramolecular interaction includes the formation of a hairpin having a stem between a universal segment at one end of the tagged target nucleic acid and a second universal segment at the opposite end of the tagged target nucleic acid. A variety of detection formats are provided, including solution-phase and surface-based formats. The methods and compositions are well-suited for highly multiplexed nucleic acid detection, and are applicable for the detection of any target nucleic acid of interest in both research and clinical settings.
19 Citations
36 Claims
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1. A method of detecting the presence or amount of a first target nucleic acid in a sample, the method comprising:
-
(a) linking first and second universal DNA segments into a first molecule in a linking reaction dependent on the first target nucleic acid, thereby providing a first tagged target nucleic acid; and (b) PCR amplifying the first tagged target nucleic acid using first and second universal primers, each universal primer having a 3′
portion that anneals to one of the two universal DNA segments;wherein;
the 3′
portion of the first universal primer anneals to the first universal segment;a 5′
portion of the first universal primer comprises a nucleic acid sequence sufficiently similar to a portion of the first universal DNA segment, a portion of the second universal DNA segment, or a portion of the first target nucleic acid, to anneal to a complement of the portion of the first universal DNA segment, a complement of the portion of the second universal DNA segment, or a complement of the portion of the first target nucleic acid, respectively, under PCR reaction conditions;an amplicon generated upon extension of the first universal primer forms an intramolecular hairpin stem between the 5′
portion of the first universal primer and a portion of the amplicon complementary to the first universal DNA segment, the second universal DNA segment, or the first target nucleic acid;
or wherein the first universal primer forms a circular structure upon annealing to the first tagged nucleic acid that brings the 3′ and
5′
-ends of the first universal primer close to each other; and
,formation of the hairpin stem or circular structure results or causes a change in a first detectable signal, the first detectable signal indicating the presence or quantity of the first target nucleic acid in the sample. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36)
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Specification