Use of products of PCR amplification carrying elements of secondary structure to improve PCR-based nucleic acid detection
First Claim
1. A method for the detection of a target nucleic acid in a sample, comprising:
- amplifying, in a reaction mixture, a target nucleic acid using PCR in the presence of a pair of oligonucleotide primers wherein at least one of the oligonucleotide primers is designed to incorporate a 5′
-specialty sequence to provide an amplification product that intramolecularly folds into a secondary structure;
and detecting the amplification product that folds into a secondary structure by a method comprising;
providing an oligonucleotide cleavage component that has no or reduced priming ability, hybridizing said oligonucleotide cleavage component with the amplification product to form a three-strand cleavage structure wherein two strands of the three-strand cleavage structure are provided by the secondary structure of the amplification product, cleaving 3′
- or 5′
-strands of the three-strand cleavage structure using a duplex-specific nuclease activity resulting in a cleavage product, and detecting the cleavage product, wherein the presence of the cleavage product is indicative of the presence of the target nucleic acid in said sample.
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Abstract
Particular aspects comprise amplifying target nucleic acid using PCR and an oligonucleotide primer pair wherein at least one of the primers is designed to incorporate a 5′-specialty sequence to provide for an amplification product that intramolecularly folds into a secondary structure; and detecting the amplification product by a method comprising: providing an oligonucleotide cleavage component, hybridizing the oligonucleotide cleavage component with the amplification product to form a three-strand cleavage structure wherein two strands of the three-strand cleavage structure are provided by the secondary structure of the amplification product, cleaving 3′- or 5′-strands of the three-strand cleavage structure using a duplex-specific nuclease activity resulting in a cleavage product, and detecting the cleavage product indicative of the presence of the target nucleic acid. In certain aspects both primers incorporate a 5′-specialty sequence and detecting comprises cleaving 3′- or 5′-strands of a three-strand cleavage structure using duplex-specific nuclease to provide a cleavage product.
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Citations
37 Claims
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1. A method for the detection of a target nucleic acid in a sample, comprising:
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amplifying, in a reaction mixture, a target nucleic acid using PCR in the presence of a pair of oligonucleotide primers wherein at least one of the oligonucleotide primers is designed to incorporate a 5′
-specialty sequence to provide an amplification product that intramolecularly folds into a secondary structure;and detecting the amplification product that folds into a secondary structure by a method comprising;
providing an oligonucleotide cleavage component that has no or reduced priming ability, hybridizing said oligonucleotide cleavage component with the amplification product to form a three-strand cleavage structure wherein two strands of the three-strand cleavage structure are provided by the secondary structure of the amplification product, cleaving 3′
- or 5′
-strands of the three-strand cleavage structure using a duplex-specific nuclease activity resulting in a cleavage product, and detecting the cleavage product, wherein the presence of the cleavage product is indicative of the presence of the target nucleic acid in said 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, 37)
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Specification