Methods for using riboprimers for strand displacement replication of target sequences
First Claim
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1. A method for amplifying a target nucleic acid sequence comprising a target nucleic acid, the method comprising the steps of:
- a) hybridizing a riboprimer to a DNA template comprising the target nucleic acid sequence, wherein said riboprimer comprises;
i) only ribonucleotides, or ii) only purine ribonucleotides and only pyrimidine nucleotides, wherein at least one of the pyrimidine nucleotides is a pyrimidine 2′
-deoxyribonucleotide having a non-canonical substituent, which substituent is neither an H nor an OH, on the 2′
-position of the deoxyribose sugar moiety;
b) extending the riboprimer with a DNA polymerase that lacks 5′
-to-3′
exonuclease activity; and
c) cleaving the annealed riboprimer with an RNAse H enzyme such that another riboprimer hybridizes to the template and repeats primer extension, whereby multiple copies of the complementary sequence of the target nucleic acid sequence are produced.
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Abstract
Methods, compositions and kits for amplifying a target sequence by strand displacement replication using strand-displacing primers. The method uses primers that have only ribonucleotides or purine ribonucleotides and at least one 2′-substituted pyrimidine-2′-deoxyribonucleotide.
78 Citations
64 Claims
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1. A method for amplifying a target nucleic acid sequence comprising a target nucleic acid, the method comprising the steps of:
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a) hybridizing a riboprimer to a DNA template comprising the target nucleic acid sequence, wherein said riboprimer comprises;
i) only ribonucleotides, or ii) only purine ribonucleotides and only pyrimidine nucleotides, wherein at least one of the pyrimidine nucleotides is a pyrimidine 2′
-deoxyribonucleotide having a non-canonical substituent, which substituent is neither an H nor an OH, on the 2′
-position of the deoxyribose sugar moiety;b) extending the riboprimer with a DNA polymerase that lacks 5′
-to-3′
exonuclease activity; andc) cleaving the annealed riboprimer with an RNAse H enzyme such that another riboprimer hybridizes to the template and repeats primer extension, whereby multiple copies of the complementary sequence of the target nucleic acid sequence are produced. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)
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21. A method for amplifying a target nucleic acid sequence comprising a target nucleic acid, the method comprising:
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a) obtaining a DNA comprising a target nucleic acid sequence; b) obtaining a riboprimer, the riboprimer comprising;
i) only ribonucleotides, or ii)only purine ribonucleotides and only pyrimidine nucleotides, wherein at least one of the pyrimidine nucleotides is a pyrimidine 2′
-deoxyribonucleotide having a non-canonical substituent, which substituent is neither an H nor an OH, on the 2′
-position of the deoxyribose sugar moiety, and wherein at least the 3′
-end portion of the riboprimer is complementary to a portion of the target nucleic acid sequence;c) annealing the riboprimer to the DNA; d) obtaining a strand-displacing DNA polymerase that lacks 5′
-to-3′
exonuclease activity;e) primer extending the riboprimer annealed to the DNA with the strand-displacing DNA polymerase under strand-displacing polymerization conditions; f) obtaining a double-stranded complex comprising the DNA and a primer extension product, wherein the primer extension product comprises the riboprimer sequence in its 5′
-end portion and the target sequence in its 3′
-end portion;g) contacting the double-stranded complex with an RNase H enzyme under enzyme reaction conditions so as to release at least a portion of the riboprimer sequence in the 5′
-end portion of the primer extension product of the double-stranded complex;h) annealing a second riboprimer to the single-stranded DNA of the double-stranded complex, wherein the second riboprimer anneals to the single-stranded DNA at the position where the portion of the riboprimer sequence of the primer extension product was released; i) primer extending the second riboprimer annealed to the single-stranded DNA of the double-stranded complex with the strand-displacing DNA polymerase under strand-displacing polymerization conditions, so as to displace the first primer extension product from the double-stranded complex and obtain a second double-stranded complex comprising the single-stranded DNA and a second primer extension product; and j) obtaining the primer extension product that was displaced from the double-stranded complex as a result of extending the second riboprimer annealed to the single-stranded DNA. - View Dependent Claims (22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38)
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39. A method of generating multiple copies of a polynucleotide sequence complementary to an RNA sequence of interest, the method comprising the steps of:
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a) extending a first primer hybridized to a target RNA with an RNA-dependent DNA polymerase, wherein the first primer is a riboprimer, wherein said riboprimer comprises;
i) only ribonucleotides, or ii) only purine ribonucleotides and only pyrimidine nucleotides, wherein at least one of the pyrimidine nucleotides is a pyrimidine 2′
-deoxyribonucleotide having a non-canonical substituent, which substituent is neither an H nor an OH, on the 2′
-position of the deoxyribose sugar moiety, and whereby a complex comprising a first primer extension product and the target RNA is produced;b) cleaving RNA in the complex of step (a) with an RNase H enzyme; c) extending a second primer hybridized to the first primer extension product with a DNA-dependent DNA polymerase that lacks 5′
-to-3′
exonuclease activity, wherein said second primer is a riboprimer comprising;
i) only ribonucleotides, or ii) only purine ribonucleotides and only pyrimidine nucleotides, wherein at least one of the pyrimidine nucleotides is a pyrimidine 2′
-deoxyribonucleotide having a non-canonical substituent, which substituent is neither an H nor an OH, on the 2′
-position of the deoxyribose sugar moiety, and whereby a second primer extension product is produced to form a complex of first and second primer extension products;d) cleaving the riboprimer in the complex of first and second primer extension products with an RNase H enzyme such that a riboprimer hybridizes to the second primer extension product; and e) extending the riboprimer hybridized to the second primer extension product with a DNA-dependent DNA polymerase, whereby the first primer extension product is displaced and multiple copies of a polynucleotide sequence complementary to the RNA sequence of interest are generated. - View Dependent Claims (40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64)
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Specification