Primer for nucleic acid detection
First Claim
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1. A method of making a fluorescently-labeled sequence-specific primer, comprising:
- adding a universal nucleic acid sequence to a sequence-specific primer, thereby generating a fluorescently-labeled sequence-specific primer,wherein the universal nucleic acid sequence comprisesa 5′
end,a 3′
end; and
a fluorescently-labeled nucleotide flanked on each side by at least one nucleotide, wherein nucleotides complementary to the nucleotides flanking the fluorescently-labeled nucleotide quench a detectable signal from the label fluorophore when the fluorescently-labeled sequence-specific primer is hybridized with its complementary nucleic acid molecule and the complementary nucleic acid is synthesized,wherein the sequence-specific primer can hybridize to a target nucleic acid sequence,wherein the fluorophore can be quenched by the nucleotides complementary to the nucleotides flanking the fluorescently-labeled nucleotide, andwherein the universal nucleic acid sequence does not substantially hybridize to the target nucleic acid sequence recognized by the sequence-specific primer.
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Abstract
This application provides universal labeled primers for detection and amplification of nucleic acid molecules. These universal primers can be attached to the 5′-end of a target sequence-specific primer. In particular examples, the universal primer includes a labeled nucleotide flanked on both sides a nucleotide whose complement nucleotides changes a detectable signal from the label when the universal primer hybridizes with its complementary nucleic acid molecule. Also disclosed are methods of using the universal primer in nucleic acid amplification, such as real-time PCR.
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Citations
25 Claims
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1. A method of making a fluorescently-labeled sequence-specific primer, comprising:
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adding a universal nucleic acid sequence to a sequence-specific primer, thereby generating a fluorescently-labeled sequence-specific primer, wherein the universal nucleic acid sequence comprises a 5′
end,a 3′
end; anda fluorescently-labeled nucleotide flanked on each side by at least one nucleotide, wherein nucleotides complementary to the nucleotides flanking the fluorescently-labeled nucleotide quench a detectable signal from the label fluorophore when the fluorescently-labeled sequence-specific primer is hybridized with its complementary nucleic acid molecule and the complementary nucleic acid is synthesized, wherein the sequence-specific primer can hybridize to a target nucleic acid sequence, wherein the fluorophore can be quenched by the nucleotides complementary to the nucleotides flanking the fluorescently-labeled nucleotide, and wherein the universal nucleic acid sequence does not substantially hybridize to the target nucleic acid sequence recognized by the sequence-specific primer. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22)
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23. A method of making a fluorescently-labeled sequence-specific primer, comprising:
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adding a fluorescently-labeled probe sequence to a sequence-specific primer, thereby generating a fluorescently-labeled sequence-specific primer, wherein the sequence-specific primer can hybridize to a target nucleic acid sequence, wherein the fluorescently-labeled probe sequence added to the sequence-specific primer comprises 5′
-CTCSSSSX(n)RRRRGAG-3′
(SEQ ID NO;
31),wherein X(n) is any number of nucleotides, wherein SSSS is complementary to RRRR, and each S is G or C, wherein T is a fluorescently-labeled nucleotide and is the second nucleotide from the end of the fluorescently-labeled probe sequence, wherein the CTCSSSS and RRRRGAG sequences of the fluorescently-labeled probe sequence are capable of hybridizing to each other, thereby quenching fluorescence from the fluorescently-labeled T, wherein the X(n) sequence is capable of hybridizing to a sequence that would be synthesized by extending the sequence-specific primer on the target nucleic acid sequence, thereby disrupting hybridization between the CTCSSSS and RRRGAG of the probe sequence, thereby increasing the fluorescent signal of the fluorescently-labeled T of the probe sequence. - View Dependent Claims (24, 25)
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Specification