Nucleic acid hairpin probes and uses thereof
First Claim
1. An oligonucleotide probe for hybridization analysis, which probe comprises a nucleotide sequence that forms a hairpin structure having a double stranded segment and a single stranded loop, wherein said loop contains at least 3 nucleotides, said double stranded segment is formed between two complementary nucleotide sequences under suitable conditions, and wherein at least a portion of said nucleotide sequences located within said double stranded segment and a portion of said nucleotide sequence located within said single stranded loop collectively form a region that is complementary to a target nucleotide sequence to be hybridized with.
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Abstract
This invention relates generally to nucleic acid hybridization analysis. More specifically, an oligonucleotide probe for hybridization analysis is provided, which probe comprises a nucleotide sequence that forms a hairpin structure having a double stranded segment and a single stranded loop, wherein at least a portion of said nucleotide sequences located within said double stranded segment and a portion of said nucleotide sequence located within said single stranded loop collectively form a region that is complementary to a target nucleotide sequence to be hybridized with. Arrays comprising the hairpin probes immobilized on a solid support and methods for nucleic acid hybridization analysis using the probes or array of immobilized probes are also provided. Methods for transcribing and/or amplifying a probe DNA sequence using a hairpin probe are further provided.
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Citations
62 Claims
- 1. An oligonucleotide probe for hybridization analysis, which probe comprises a nucleotide sequence that forms a hairpin structure having a double stranded segment and a single stranded loop, wherein said loop contains at least 3 nucleotides, said double stranded segment is formed between two complementary nucleotide sequences under suitable conditions, and wherein at least a portion of said nucleotide sequences located within said double stranded segment and a portion of said nucleotide sequence located within said single stranded loop collectively form a region that is complementary to a target nucleotide sequence to be hybridized with.
- 16. An array of oligonucleotide probes immobilized on a solid support for hybridization analysis, which array comprises a solid support suitable for use in nucleic acid hybridization having immobilized thereon a plurality of oligonucleotide probes, at least one of the probes comprises a nucleotide sequence that forms a hairpin structure having a double stranded segment and a single stranded loop, wherein said loop contains at least 3 nucleotides, said double stranded segment is formed between two complementary nucleotide sequences under suitable conditions, and wherein at least a portion of said nucleotide sequences located within said double stranded segment and a portion of said nucleotide sequence located within said single stranded loop collectively form a region that is complementary to a target nucleotide sequence to be hybridized with.
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32. A method for detecting a target nucleotide sequence in a sample, which method comprises the steps of:
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a) providing an oligonucleotide probe comprising a nucleotide sequence that forms a hairpin structure having a double stranded segment and a single stranded loop, wherein said loop contains at least 3 nucleotides, said double stranded segment is formed between two complementary nucleotide sequences under suitable conditions, and wherein at least a portion of said nucleotide sequences located within said double stranded segment and a portion of said nucleotide sequence located within said single stranded loop collectively form a region that is complementary to a target nucleotide sequence to be detected;
b) contacting the probe provided in step a) with a sample containing or suspected of containing the target nucleotide sequence under conditions that favor intermolecular hybridization between the probe and the target nucleotide sequence over intramolecular hybridization of the probe itself; and
c) assessing the intermolecular hybrid formed in step b). - View Dependent Claims (33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58)
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59. A method for transcribing and/or amplifying an oligonucleotide probe sequence, which method comprises the steps of:
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a) providing an oligonucleotide probe comprising a nucleotide sequence that forms a hairpin structure having a double stranded segment and a single stranded loop, wherein said loop contains at least 3 nucleotides, said double stranded segment is formed between two complementary nucleotide sequences under suitable conditions and contains a promoter sequence, and wherein at least a portion of said nucleotide sequence located within said single stranded loop is complementary to a DNA sequence and said portion of said nucleotide sequence comprises both ribonucleotide sequence and deoxyribonucleotide sequence;
b) contacting said probe provided in step a) with said DNA sequence under suitable conditions to form a probe/DNA duplex;
c) cleaving said ribonucleotide sequence within said portion of said nucleotide sequence complementary to said DNA sequence by RNase H treatment to open said single stranded loop; and
d) synthesizing a RNA sequence using a RNA polymerase that is compatible with said promoter contained within said double stranded segment of said probe, whereby at least a portion of said single stranded loop is transcribed. - View Dependent Claims (60, 61, 62)
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Specification