Rnase h-based assays utilizing modified rna monomers

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.

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.   
     
     
         2 . The method of  claim 1 , wherein the hot start cleaving enzyme is an RNase H enzyme. 
     
     
         3 . The method of  claim 2 , wherein said RNase H enzyme is an RNase H2 enzyme. 
     
     
         4 . The method of  claim 3 , wherein said RNase H2 enzyme inherently has lower activity at reduced temperature, is reversibly inactivated by chemical modification or by a blocking antibody. 
     
     
         5 . The method of  claim 1 , wherein said cleaving enzyme is a sequence specific double stranded endonuclease. 
     
     
         6 . The method of  claim 5 , wherein said sequence specific double stranded endonuclease is a restriction enzyme. 
     
     
         7 . The method of  claim 1 , wherein the blocking group is attached to the 3′-terminal nucleotide of the primer. 
     
     
         8 . The method of  claim 1 , wherein the blocking group is attached 5′ of the 3′-terminal residue. 
     
     
         9 . The method of  claim 8 , wherein the blocking group includes one or more abasic residues or modified nucleosides. 
     
     
         10 . The method of  claim 9 , wherein the abasic residue is a C3 spacer. 
     
     
         11 . The method of  claim 9 , wherein the modified nucleoside is a 2′-O-methyl ribose residue. 
     
     
         12 . The method of  claim 1 , wherein the blocking group includes a label permitting detection of the amplification reaction. 
     
     
         13 . The method of  claim 12 , wherein the label is a fluorophore, a quencher, biotin, or a hapten. 
     
     
         14 . The method of  claim 12 , wherein the label is a mass tag for detection of the amplification reaction by mass spectrometry. 
     
     
         15 . The method of  claim 2 , wherein the cleavage domain is a continuous sequence of 3 or more RNA residues. 
     
     
         16 . The method of  claim 15 , wherein said cleavage domain further comprises one or more of the following moieties: a DNA residue, an abasic residue, a modified nucleoside, or a modified phosphate internucleotide linkage. 
     
     
         17 . The method of  claim 3 , wherein the cleavage domain is a single RNA residue or two adjacent RNA residues. 
     
     
         18 . The method of  claim 3 , wherein the cleavage domain lacks an RNA residue. 
     
     
         19 . The method of  claim 18 , wherein the cleavage domain comprises one or more 2′-modified nucleosides. 
     
     
         20 . The method of  claim 19 , wherein said 2′-modified nucleoside is a single 2′-fluoronucleoside. 
     
     
         21 . The method of embodiment 19, wherein the cleavage domain is two adjacent 2′-fluoronucleoside residues. 
     
     
         22 . The method of  claim 18 , wherein the cleavage reaction is carried out in the presence of one or more of the following divalent cations: manganese, cobalt, nickel or zinc. 
     
     
         23 . The method of  claim 22 , wherein magnesium is also present in the reaction mixture. 
     
     
         24 . The method of  claim 17 , wherein a sequence within or flanking the cleavage domain contains one or more internucleoside linkages resistant to nuclease cleavage. 
     
     
         25 . The method of  claim 24 , wherein said nuclease resistant linkage is phosphorothioate, phosphorodithioate, methylphosphonate or an abasic residue. 
     
     
         26 . The method of  claim 24 , wherein said nuclease resistant linkage is on the 3′ side of the cleavage domain. 
     
     
         27 . The method of  claim 1 , further comprising a second primer in reverse orientation from the first primer to support PCR. 
     
     
         28 . The method of  claim 27 , wherein the second primer is an unmodified DNA primer. 
     
     
         29 . The method of  claim 27 , wherein the second primer comprises a cleavage domain (new) 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. 
     
     
         30 . The method of  claim 27 , wherein the PCR assay is used to discriminate between variant alleles. 
     
     
         31 . The method of  claim 30 , wherein a secondary mutation site is incorporated within or flanking the cleavage domain to enhance detection of the variant allele. 
     
     
         32 . The method of  claim 30 , wherein a modified nucleoside is incorporated within or flanking the cleavage domain to enhance detection of the variant allele. 
     
     
         33 . The method of  claim 32 , wherein said modified nucleoside is a 2′-O-methyl ribose residue. 
     
     
         34 . The method of  claim 30 , wherein a nuclease resistant linkage is incorporated on the 3′-side of the cleavage domain. 
     
     
         35 . The method of  claim 27 , wherein the PCR assay is used to quantitate the abundance of the target nucleic sequence in the sample. 
     
     
         36 . The method of embodiment 27, wherein the PCR assay is a primer-probe PCR assay. 
     
     
         37 . The method of  claim 36 , wherein the primer having a 5′ label domain includes a cleavage domain. 
     
     
         38 . The method of  claim 37 , wherein the cleavage domain is an RNase H cleavage domain. 
     
     
         39 . The method of  claim 38 , wherein the RNase H cleavage domain is an RNase H2 cleavage domain. 
     
     
         40 . 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 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.   
     
     
         41 . The method of  claim 40  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. 
     
     
         42 . The method of  claim 41  wherein said cleavage domain has the sequence RDxxD. 
     
     
         43 . The method of  claim 41  wherein x is a C3 spacer. 
     
     
         44 . The method of  claim 40  wherein the assay is used to discriminate between variant alleles. 
     
     
         45 . The method of  claim 44  wherein said allelic variants are single nucleotide polymorphisms.