RNASE H-BASED ASSAYS UTILIZING MODIFIED RNA MONOMERS
First Claim
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1. A method of amplifying a target DNA sequence, said method comprising the steps of:
- (a) providing a reaction mixture comprising(i) an oligonucleotide primer having a cleavage domain positioned 5′
of a blocking group, said blocking group linked at or near the end of the 3′
-end of the oligonucleotide primer wherein said blocking group prevents primer extension and/or inhibits the primer from serving as a template for DNA synthesis,(ii) a sample nucleic acid that may or may not have the target sequence,(iii) a cleaving enzyme and(iv) a polymerase wherein said cleaving enzyme is a hot start cleaving enzyme which is thermostable and has reduced activity at lower temperatures;
(b) hybridizing the primer to the target DNA sequence to form a double-stranded substrate;
(c) cleaving the hybridized primer with said cleaving enzyme at a point within or adjacent to the cleavage domain to remove the blocking group from the primer; and
(d) extending the primer with the polymerase.
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Abstract
The present invention pertains to novel oligonucleotide compounds for use in various biological assays, such as nucleic acid amplification, ligation and sequencing reactions. The novel oligonucleotides comprise a ribonucleic acid domain and a blocking group at or near the 3′ end of the oligonucleotide. These compounds offer an added level of specificity previously unseen. Methods for performing nucleic acid amplification, ligation and sequencing are also provided. Additionally, kits containing the oligonucleotides are also disclosed herein.
45 Citations
45 Claims
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1. A method of amplifying a target DNA sequence, said method comprising the steps of:
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(a) providing a reaction mixture comprising (i) an oligonucleotide primer having a cleavage domain positioned 5′
of a blocking group, said blocking group linked at or near the end of the 3′
-end of the oligonucleotide primer wherein said blocking group prevents primer extension and/or inhibits the primer from serving as a template for DNA synthesis,(ii) a sample nucleic acid that may or may not have the target sequence, (iii) a cleaving enzyme and (iv) a polymerase wherein said cleaving enzyme is a hot start cleaving enzyme which is thermostable and has reduced activity at lower temperatures; (b) hybridizing the primer to the target DNA sequence to form a double-stranded substrate; (c) cleaving the hybridized primer with said cleaving enzyme at a point within or adjacent to the cleavage domain to remove the blocking group from the primer; and (d) extending the primer with the polymerase. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35)
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21. The method of embodiment 19, wherein the cleavage domain is two adjacent 2′
- -fluoronucleoside residues.
- 36. The method of embodiment 27, wherein the PCR assay is a primer-probe PCR assay.
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40. A method of amplifying a target DNA sequence, said method comprising the steps of:
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(a) providing a reaction mixture comprising (i) an oligonucleotide primer having a cleavage domain with a cleavage site which is cleavable by an RNase H2 enzyme, wherein said cleavage site is positioned 5′
of a blocking group at or near the 3′
-end of the oligonucleotide primer which prevents primer extension and/or PCR, and wherein said cleavage domain includes an RNA residue and an abasic residue,(ii) a sample nucleic acid that may or may not have the target sequence, (iii) a DNA polymerase, (iv) an RNase H2 enzyme; and (v) optionally a second primer in reverse orientation to support PCR; (b) hybridizing the blocked primer to the target DNA sequence in said reaction mixture to form a double-stranded substrate; (c) cleaving the hybridized blocked primer with said RNase H2 to remove the blocking group from the primer; and (d) extending the cleaved primer with the DNA polymerase. - View Dependent Claims (41, 42, 43, 44, 45)
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Specification