RNase H-based assays utilizing modified RNA monomers
AssigneeIntegrated Dna Technologies, Inc.
InventorsJoseph Alan Walder, Mark Aaron Behlke, Scott Rose, Joseph Dobosy
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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.
Claims28
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 with a cleavage site, located within or adjacent to the cleavage domain, 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,
- (ii) a sample nucleic acid that may or may not have the target sequence,
- (iii) a DNA polymerase,
- (iv) an RNase H2 enzyme encoded by SEQ ID NO:4, and
- (v) optionally a second primer in reverse orientation to support PCR;
- (b) hybridizing the blocked primer to the target DNA sequence to form a double-stranded substrate;
- (c) cleaving the hybridized blocked primer with said RNase H2 enzyme to remove the blocking group from the primer;
- and
- (d) extending the cleaved primer with the DNA polymerase.
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 with a cleavage site, located within or adjacent to the cleavage domain, 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 a sequence 5′ to 3′ Rx or RDx where R is an RNA residue, D is a DNA residue and x is 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.
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 with a cleavage site, located within or adjacent to the cleavage domain, 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 lacks an RNA residue and comprises one or more 2'-modified nucleosides,
- (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 enzyme to remove the blocking group from the primer;
- and
- (d) extending the cleaved primer with the DNA polymerase.
Dependencies are inferred from the claim text.