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 substantially identical 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;
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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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.
71 Citations
53 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:
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(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 substantially identical 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;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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37. An analyte nucleic acid detection reaction mixture, comprising:
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an analyte nucleic acid comprising a nucleic acid subsequence of interest, the analyte nucleic acid further comprising first, second and third tag sequences, the second tag sequence being located between the first and third tag sequences; a first universal primer comprising a first tag complement subsequence that is complementary to the first tag sequence, a subsequence that comprises the second tag sequence, or a subsequence thereof, and a detectable label; and
,a second universal primer comprising a sequence that comprises the third tag sequence or a subsequence thereof. - View Dependent Claims (38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49)
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50. A method of detecting an analyte nucleic acid, the method comprising:
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(a) providing a reaction mixture comprising; (i) an analyte nucleic acid comprising a nucleic acid subsequence of interest, the analyte nucleic acid further comprising first, second and third tag sequences, the second tag sequence being located between the first and third tag sequences; (ii) a first universal primer comprising a first tag complement subsequence that is complementary to the first tag sequence, a subsequence that comprises the second tag sequence, or a subsequence thereof, a label, and a label quencher; and (iii) a second universal primer comprising a sequence that comprises the third tag sequence or a subsequence thereof; (b) annealing the tag complement subsequence of the first universal primer to the first tag of the analyte nucleic acid; (c) performing a first primer extension reaction that extends the first universal primer, wherein a hairpin stem forms between first and second portions of the product of the first primer extension reaction, wherein the first portion comprises the second tag sequence, and wherein the second portion is complementary to the second tag sequence; (d) annealing the second universal primer to the product of the first primer extension reaction; and (e) performing a second primer extension reaction that extends the universal specificity primer, wherein the second primer extension reaction releases the label and/or label quencher from the product of the first primer extension reaction; and
,(f) detecting the label, which indicates the presence of the analyte nucleic acid. - View Dependent Claims (51, 52, 53)
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Specification