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, (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, a 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  claim 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 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  claim 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 positioned 5′ of a blocking group, said blocking group linked upstream 5′ of the 3′-terminal residue of the oligonucleotide primer wherein said blocking group prevents primer extension, (ii) a sample nucleic acid that may or may not have the target sequence, (iii) a cleaving enzyme and (iv) a polymerase;   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.   
     
     
         41 . A primer for DNA replication having a 5′ end and a 3′ end, said primer comprising:
 a) a nonactivated configuration when the primer is unhybridized to a DNA sequence of interest, the nonactivated configuration further comprising an RNase H cleavage domain comprising one or more 2′-modified nucleoside residues, and a blocking group linked at or near to the 3′ end of the primer;   b) an activated configuration when the primer is hybridized to the DNA sequence of interest, wherein the activated configuration is the free of the 3′ blocking group and is capable of supporting DNA replication.   
     
     
         42 . The composition of  claim 41  wherein said 2′-modified nucleoside residue is a 2′-fluoronucleoside. 
     
     
         43 . A primer for DNA replication having a 5′ end and a 3′ end, said primer comprising:
 a) a nonactivated configuration when the primer is unhybridized to a DNA sequence of interest, the nonactivated configuration further comprising an RNase H cleavage domain, and a blocking group linked at or near to the 3′ end of the primer which incorporates a label;   b) an activated configuration when the primer is hybridized to the DNA sequence of interest, wherein the activated configuration is the free of the 3′ blocking group and is capable of supporting DNA replication.   
     
     
         44 . A primer for DNA replication having a 5′ end and a 3′ end, said primer comprising:
 a) a nonactivated configuration when the primer is unhybridized to a DNA sequence of interest, the nonactivated configuration further comprising an RNase H cleavage domain, and a blocking group located 5′ to the 3′-terminal nucleotide residue of the primer;   b) an activated configuration when the primer is hybridized to the DNA sequence of interest, wherein the activated configuration is the free of the 3′ blocking group and is capable of supporting DNA replication.   
     
     
         45 . A method of detection of a target nucleic acid sequence within a sample using a cycling probe reaction, wherein cleavage of the probe is mediated by a thermophilic RNAse H2 enzyme and a cleavage domain lacks an RNA residue. 
     
     
         46 . The method of  claim 45  wherein the cleavage domain comprises one or more 2′-modified nucleoside residues. 
     
     
         47 . The method of  claim 46  wherein the cleavage domain is comprised of one or more 2′-nucleoside residues. 
     
     
         48 . A method of detection of a target nucleic acid sequence within a sample using a cycling probe reaction, wherein cleavage of the probe is mediated by a thermophilic hot start RNAse H2 enzyme having reduced activity at lower temperatures. 
     
     
         49 . A method of detection of a target nucleic acid sequence within a sample using an oligonucleotide ligation reaction, wherein said method comprises the steps of:
 a) contacting the sample with an acceptor oligonucleotide comprising a cleavage domain and a blocking group at or near the 3′-end wherein said blocking group inhibits ligation;   b) hybridizing the acceptor oligonucleotide to the target DNA sequence to form a double-stranded substrate;   c) cleaving said hybridized acceptor of the oligonucleotide with a cleaving enzyme at a point within or adjacent to the cleavage domain to remove the blocking group; and   d) ligating the acceptor oligonucleotide to a donor oligonucleotide with a ligase.   
     
     
         50 . The method of  49  wherein said cleavage domain is an RNase H2 cleavage domain. 
     
     
         51 . The method of  claim 49  wherein the oligonucleotide ligation assay is used to distinguish allelic variants. 
     
     
         52 . The method of  claim 49  wherein said allelic variants are single nucleotide polymorphisms. 
     
     
         53 . A method of detection of a target nucleic acid sequence within a sample using an oligonucleotide ligation reaction, wherein said method comprises the steps of:
 a) contacting the sample with an donor oligonucleotide comprising a cleavage domain and a blocking group at or near the 5′-end wherein said blocking group inhibits ligation;   b) hybridizing the donor oligonucleotide to the target DNA sequence to form a double-stranded substrate;   c) cleaving said hybridized donor oligonucleotide with a cleaving enzyme at a point within or adjacent to the cleavage domain to remove the blocking group; and   d) ligating the donor oligonucleotide to an acceptor oligonucleotide with a ligase.   
     
     
         54 . The method of  53  wherein said cleavage domain is an RNase H2 cleavage domain. 
     
     
         55 . A method of sequencing a target nucleic acid comprising the steps of:
 a) providing a reaction mixture comprising the target nucleic acid, an acceptor oligonucleotide, a donor oligonucleotide set, the donor oligonucleotide set comprising four oligonucleotide groups corresponding to guanine, cytosine, adenine and thymine nucleotides, wherein each oligonucleotide group comprises donor oligonucleotides that are about 7 to 11 bases long, wherein a first domain on the 5′ end of the donor oligonucleotide comprises one or two bases and correspond to the nucleotide or nucleotides of the given set, a second domain 3′ to the first domain cleavable by an RNase H2 enzyme, a third domain 3′ to the second domain comprising degenerate or universal bases, and a fourth domain comprising a label and a blocking group at or near the 3′-end, which can be the label, of the donor oligonucleotide preventing it from serving as an acceptor in a ligation reaction;   b) hybridizing the acceptor oligonucleotide to the target nucleic acid sequence;   c) hybridizing the donor oligonucleotides to the target nucleic acid sequence;   d) introducing a ligase enzyme to ligate the donor oligonucleotide and the acceptor oligonucleotide;   e) detection of the label on the donor oligonucleotide ligated to the acceptor;   f) introducing an RNase H2 enzyme to cleave the donor oligonucleotide 5′ of the RNA or modified residue and leaving the first domain of the donor molecule attached to the acceptor oligonucleotide; and   g) repeating steps c-f.   
     
     
         56 . A method of synthesizing a nucleic acid, comprising the steps of:
 a) providing a reaction mixture comprising an acceptor oligonucleotide, a donor oligonucleotide, said donor oligonucleotide having a 5′-phosphate and comprising a specific base at the 5′-end, an adjacent RNA residue, a hairpin structure with a 3′-overhang of 1 to 8 residues, said overhang comprising degenerate or universal bases, optionally a blocking group at or near the 3′-end and a label;   b) hybridizing the acceptor oligonucleotide to the 3′-overhanf of the donor oligonucleotide;   c) introducing a ligase enzyme to ligate the donor oligonucleotide and the acceptor oligonucleotides;   d) introducing an RNase H2 enzyme to cleave the donor oligonucleotide 5′ of the RNA residue and leaving the first base on the 5′ end of the donor molecule attached to the acceptor oligonucleotide; and   e) repeating steps b-d.   
     
     
         57 . A method for determining a DNA sequence, said method comprising the steps of:
 a) providing a reaction mixture comprising 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, a sample nucleic acid, a cleaving enzyme, a polymerase, labeled dideoxynucleotide triphosphates and unlabeled deoxynucleotide triphosphates;   b) hybridizing the primer to the 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; and   e) separating the 5′-nested fragments formed in the extension reaction.   
     
     
         58 . A method for determining a DNA sequence, said method comprising the steps of:
 a) providing a reaction mixture comprising a fluorescently labeled oligonucleotide primer, a sample nucleic acid, a polymerase, ribonucleotide triphosphates, and deoxyribonucleotide triphosphates;   b) hybridizing the primer to the sample DNA sequence to form a double-stranded substrate;   c) extending the primer with the polymerase;   d) cleaving the double stranded product of the extension reaction with an RNase H2 enzyme; and   e) separating the 5′-nested labeled fragments formed in the extension reaction.   
     
     
         59 . A primer for DNA replication having a 5′ end and a 3′ end, said primer comprising:
 a) a nonactivated configuration when the primer is unhybridized to a DNA sequence of interest, the nonactivated configuration further comprising a thermostable RNAse H cleavage domain and blocking group linked at or near to the 3′ end of the primer;   b) an activated configuration when the primer is hybridized to the DNA sequence of interest, wherein the activated configuration is the free of the 3′ blocking group and is capable of supporting DNA replication.   
     
     
         60 . A single stranded oligonucleotide having a 3′ end and a 5′ end, the single stranded oligonucleotide comprising a ribonucleic acid domain comprising at least one RNA residue, and a blocking group linked at or near to the 3′ end of the oligonucleotide. 
     
     
         61 . The single stranded oligonucleotide of  claim 60 , wherein the ribonucleic acid moiety consists of 1 to 3 ribonucleic acid bases, or modified derivatives thereof. 
     
     
         62 . The single stranded oligonucleotide of  claim 60 , wherein the ribonucleic acid domain is located at least 3 bases from the 3′ end of the compound. 
     
     
         63 . The single stranded oligonucleotide of  claim 60 , wherein the ribonucleic acid domain is located at least 5 bases from the 3′ end of the compound. 
     
     
         64 . The single stranded oligonucleotide of  claim 60 , wherein the ribonucleic acid domain is located at the 3′ end of the compound. 
     
     
         65 . The single stranded oligonucleotide of  claim 60 , wherein the ribonucleic acid domain comprises 1 or 2 RNA bases. 
     
     
         66 . The single stranded oligonucleotide of  claim 60 , wherein the ribonucleic acid domain comprises one RNA base. 
     
     
         67 . The single stranded oligonucleotide of  claim 60 , wherein at least one RNA residue contains one or more modifications. 
     
     
         68 . The single stranded oligonucleotide of  claim 67 , wherein the modification confers resistance to cleavage by single-stranded ribonucleases and water catalyzed hydrolysis. 
     
     
         69 . The single stranded oligonucleotide of  claim 67 , wherein the modification is at the 2′ position of the RNA residue. 
     
     
         70 . The single stranded oligonucleotide of  claim 69 , wherein the modification is selected from the group consisting of 2′ fluoro, 2′ alkyl, 2′ methyl, 2′ amino, 2′ LNA, 2′ ENA, 2′ thio, 2′-O-alkyl, 2′-O-methyl, 5′ thio, 5′ amine, 5′ alkyl, 5′ methylene, 5′ ethylene, 3′-phosphate and 3′-phosphate diester. 
     
     
         71 . The single stranded oligonucleotide of  claim 67 , wherein the modifications are at the phosphate group on the 3′ side of the RNA base. 
     
     
         72 . The single stranded oligonucleotide of  claim 67 , wherein the modifications are at the 5′ position on the adjacent residue on the 3′ side of the RNA base. 
     
     
         73 . The single stranded oligonucleotide of  claim 67 , wherein the modifications are selected from the group consisting of phosphorothioate, phosphorodithioates and boronate. 
     
     
         74 . The single stranded oligonucleotide of  claim 73 , wherein the modifications are selected from the group consisting of 5′ thio, 5′ amine and 5′ alkyl, 5′ methylene and 5′ ethylene. 
     
     
         75 . The single stranded oligonucleotide of  claim 60 , wherein the blocking group is selected from the group consisting of a dideoxy nucleotide, 3′ hydroxyl modified DNA residue, 2′ hydroxyl substitutions or any substitution off of the base group of a monomer at or near the 3′ end capable of preventing extension or ligation. 
     
     
         76 . The single stranded oligonucleotide of  claim 75 , wherein the 3′ hydroxyl modified DNA residues are selected from the group consisting of dideoxy, 3′-phosphate and 3′-phosphate diester. 
     
     
         77 . The single stranded oligonucleotide of  claim 76 , wherein the 3′ phosphate diester is 3′-hydroxypropyl diester. 
     
     
         78 . The single stranded oligonucleotide of  claim 75 , wherein the 2′ hydroxyl substitutions are selected from the group consisting of triisopropyl silyl and tert-butyl dimethyl silyl. 
     
     
         79 . The single stranded oligonucleotide of  claim 67 , wherein the one or more RNA bases or modified residues serves as a substrate for RNase H2 are located at least 8 bases away from the 3′ end and/or the 5′ end of the primer. 
     
     
         80 . An oligonucleotide primer for DNA replication having a 5′ end and a 3′ end comprising:
 a) a 5′ sequence tag that is not complementary to the target nucleic acid;   b) a first cleavage domain located within or 3′ to the 5′ sequence tag and which is cleavable when hybridized to a complementary base or sequence;   c) a target hybridizing sequence located 3′ to the 5′ sequence tag and the first cleaving domain, wherein the target hybridizing sequence is at least partially complementary to a target nucleic acid;   d) a blocking group located 3′ to the hybridizing sequence, the blocking group preventing extension of the primer;   e) a second cleavage domain located between the target hybridizing sequence and the blocking group.   
     
     
         81 . The primer of  claim 80 , wherein the second cleavage domain will be cleaved at a higher rate when the second cleaving domain is complementary to the target nucleic acid. 
     
     
         82 . A double stranded nucleic acid compound comprising:
 a) a first single stranded oligonucleotide having a 3′ end and a 5′ end, a ribonucleic acid domain comprising at least one RNA residue, and a blocking group linked at or near the 3′ end of the oligonucleotide; and   b) a second single stranded oligonucleotide having a 3′ end and a 5′ end, a region substantially complimentary to the first single stranded oligonucleotide, and a target DNA sequence.   
     
     
         83 . A single stranded oligonucleotide probe having a 3′ end and a 5′ end an RNase H cleavage domain comprising one or more modified nucleotide residues, a fluorophore, and a quencher, wherein the RNase H cleavage domain is positioned between the fluorophore and the quencher. 
     
     
         84 . The single stranded oligonucleotide probe of  claim 83 , wherein the modified nucleotide residues are selected from the group consisting of 2′ fluoro, 2′ alkyl, 2′ methyl, 2′ amino, 2′ LNA, 2′ ENA, 2′ thio, 2′-O-alkyl, 2′-O-methyl, 5′ thio, 5′ amine, 5′ alkyl, 5′ methylene, 5′ ethylene, 3′-phosphate and 3′-phosphate diester. 
     
     
         85 . A method for replicating a target nucleic acid sequence comprising the steps of:
 a) providing a reaction mixture comprising at least one oligonucleotide primer having a blocking group at or near the 3′ end, and an RNase H2 cleavage domain 5′ to the blocking group, a target nucleic acid, an RNaseH2 enzyme and a polymerase;   b) hybridizing the at least one primer to the target nucleic acid to form a double stranded substrate;   c) removing the blocking group with the RNaseH2 enzyme; and   d) extending the primer oligonucleotide with the polymerase.   
     
     
         86 . The method of  claim 85 , wherein the reaction mixture contains at least two oligonucleotide primers having a blocking group at or near the 3′ end, and an RNase H cleavage domain 5′ to the blocking group. 
     
     
         87 . The method of  claim 85 , wherein the RNaseH2 enzyme has a 10-fold increase in activity at 75° C. compared to an activity level at 25° C. 
     
     
         88 . The method of  claim 85 , wherein the blocking group is a capped 3′ nucleic acid residue. 
     
     
         89 . The method of  claim 85 , wherein the blocking group is between the RNase H2 cleaving domain and the 3′ end. 
     
     
         90 . The method of  claim 89 , wherein the blocking group is a C3 spacer. 
     
     
         91 . The method of  claim 85 , wherein one or more C3 spacers are located between the RNase H2 cleaving domain and the 3′ end, and wherein at least one of the C3 spacers act as the blocking group. 
     
     
         92 . The method of  claim 85 , wherein the RNase H2 cleavage domain comprises at least one modified oligonucleotide. 
     
     
         93 . The method of  claim 85 , wherein the RNase H2 cleavage domain comprises two adjacent 2′-fluoro nucleotides. 
     
     
         94 . A method for amplifying a target nucleic acid sequence within a nucleic acid polymer comprising the steps of:
 a) contacting the target nucleic acid sequence with a reaction mixture comprising:
 (i) a primer set containing a plurality of forward primers and a plurality of reverse primers, wherein at least one of the pluralities of forward or reverse primers comprises individual primers having a blocking group at or near the 3′ end, and an RNase H2 cleavage domain 5′ to the blocking group, (ii) an RNaseH2 enzyme, (iii) dNPTs and (iv) a nucleic acid polymerase enzyme, in which, 
   b) each of the reverse primers is capable of hybridizing to the nucleic acid polymer and forming a first primer extension product using the nucleic acid polymer as a template, the first primer extension product comprising the reverse primer sequence and structure and at least a portion of the target nucleic acid sequence or its complement; and   c) each of the forward primers is capable of hybridizing to the first primer extension product and forming a second primer extension product using the first primer extension product as a template;   d) the plurality of primers containing the blocking group at or near the 3′end, and an RNase H2 cleavage domain 5′ to the blocking group will not extend until the hybridized to a complementary sequence and the cleavage domain cleaved by the RNaseH2 enzyme;   e) subjecting the reaction mixture to conditions such that
 (i) the reverse primer hybridizes to the nucleic acid polymer and forms the first primer extension product, and (ii) the forward primer hybridizes to the first primer extension product to form the second primer extension product; 
   f) separating the first and second primer extension products from the nucleic acid polymer templates and treating the resulting mixture with the primer set under conditions such that additional first and second primer extension products are produced; and   g) repeating step (f) to provide a reaction mixture wherein the at least a portion of the nucleotide sequence of interest or its complement is amplified.   
     
     
         95 . The method of  claim 94  wherein the RNase H2 cleavage domain is located at least 3 bases from the 3′ end of the compound. 
     
     
         96 . The method of  claim 94 , wherein the RNase H2 cleavage domain is located at least 5 bases from the 3′ end of the compound. 
     
     
         97 . The method of  claim 94 , wherein the RNase H2 cleavage domain is located at the 3′ end of the compound. 
     
     
         98 . The method of  claim 94 , wherein the RNase H2 cleavage domain comprises one RNA base. 
     
     
         99 . The method of  claim 94 , wherein the RNase H2 cleavage domain is 8 bases away from 3′ end of the primer and has at least one RNA residue or at least one modified residue serving as a substrate for RNase H2. 
     
     
         100 . The method of  claim 94 , wherein the RNase H2 enzyme has a 10-fold increase in activity at 75° C. compared to an activity level at 25° C. 
     
     
         101 . The method of  claim 94 , wherein the blocking group is a capped 3′ nucleic acid residue. 
     
     
         102 . The method of  claim 94 , wherein the blocking group is between the RNase H2 cleaving domain and the 3′ end. 
     
     
         103 . The method of  claim 102 , wherein the blocking group is a C3 spacer. 
     
     
         104 . The method of  claim 94 , wherein one or more C3 spacers are located between the RNase H2 cleaving domain and the 3′ end, and wherein at least one of the C3 spacers act as the blocking group. 
     
     
         105 . The method of  claim 94 , wherein the RNase H2 cleavage domain comprises at least one modified oligonucleotide. 
     
     
         106 . The method of  claim 94 , wherein the RNase H2 cleavage domain comprises two adjacent 2′-fluoro nucleotides. 
     
     
         107 . The method of  claim 94 , wherein individual primers in both pluralities of primers contain a blocking group at or near the 3′ end, and an RNase H cleavage domain 5′ to the blocking group. 
     
     
         108 . The method of  claim 94 , wherein amplification of the target nucleic acid is monitored by fluorescence, wherein the amount of fluorescence generated is directly proportional to the amount of target nucleic acid amplification. 
     
     
         109 . The method of  claim 94 , wherein the target nucleic acid is amplified polynomially. 
     
     
         110 . The method of  claim 94 , wherein the first primer extension product contains the cleavage domain of the reverse primer and extension of the second primer extension product terminates at the cleavage domain of the first primer extension product such that the second primer extension product does not contain a sufficiently complementary sequence to hybridize with the reverse primer and form a first primer extension product. 
     
     
         111 . The method of  claim 94 , wherein the second primer extension product contains the cleavage domain of the forward primer and extension of the first primer extension product terminates at the cleavage domain of the second primer extension product such that the first primer extension product does not contain a sufficiently complementary sequence to hybridize with the forward primer and form a second primer extension product. 
     
     
         112 . A method for quantifying the amplification of a target nucleic acid sequence within a nucleic acid polymer comprising the steps of:
 a) contacting the target nucleic acid sequence with a reaction mixture comprising:
 (i) a primer set containing a plurality of forward primers and a plurality of reverse primers, (ii) an oligonucleotide probe having a 3′ end and a 5′ end comprising an RNase H cleavage domain comprising one or more modified nucleotide residues, a fluorophore, and a quencher, wherein the RNase H cleavage domain is positioned between the fluorophore and the quencher, (iii) an RNase H enzyme, (iv) dNPTs and (v) a nucleic acid polymerase enzyme, in which, 
   b) each of the reverse primers is capable of hybridizing to the nucleic acid polymer and forming a first primer extension product using the nucleic acid polymer as a template, the first primer extension product comprising the reverse primer sequence and structure and at least a portion of the target nucleic acid sequence or its complement; and   c) each of the forward primers is capable of hybridizing to the first primer extension product and forming a second primer extension product using the first primer extension product as a template;   d) subjecting the reaction mixture to conditions such that
 (i) the reverse primer hybridizes to the nucleic acid polymer and forms the first primer extension product, (ii) the forward primer hybridizes to the first primer extension product to form the second primer extension product and, (iii) the oligonucleotide probe binds either the first or second primer extension product, and the probe is cleaved at its cleavage domain by the RNase H enzyme after hybridization to the first or second primer extension product; 
   e) measuring the fluorescence generated by cleavage of the probe;   f) separating the first and second primer extension products from the nucleic acid polymer templates and treating the resulting mixture with the primer set under conditions such that additional first and second primer extension products are produced; and   g) repeating steps (f) and (g) to provide a reaction mixture wherein the at least a portion of the nucleotide sequence of interest or its complement is amplified and the amplification is quantified by measuring the fluorescence generated by cleavage of the probe.   
     
     
         113 . The method of  claim 112 , wherein the RNase H2 cleavage domain is located at least 3 bases from the 3′ end of the compound. 
     
     
         114 . The method of  claim 112 , wherein the RNase H2 cleavage domain is located at least 5 bases from the 3′ end of the compound. 
     
     
         115 . The method of  claim 112 , wherein the RNase H2 cleavage domain is located at the 3′ end of the compound. 
     
     
         116 . The method of  claim 112 , wherein the RNase H2 cleavage domain comprises one RNA base. 
     
     
         117 . The method of  claim 112 , wherein the RNase H2 cleavage domain is 8 bases away from 3′ end of the primer and has at least one RNA residue or at least one modified residue serving as a substrate for RNase H2. 
     
     
         118 . The method of  claim 112 , wherein the RNase H2 enzyme is thermostable. 
     
     
         119 . The method of  claim 112 , wherein the oligonucleotide probe is cleaved by RNase H2. 
     
     
         120 . A method for detecting a single base mutation in a target nucleic acid sample, the method comprising the steps of:
 a) mixing a sample containing the target nucleic acid, a primer extension reaction mixture, and an RNase H2 enzyme;   b) incubating the reaction mixture with at least one primer, the primer having a 5′ end and a 3′ end, said primer comprising (i) a region at least partially complementary to the target nucleic acid and is capable of supporting DNA replication, (ii) a cleavage domain that is capable of being cleaved by the RNase H2 enzyme when the primer hybridizes to the target nucleic acid and the cleavage domain is aligned to a potential base mutation site and complementary to the potential base mutation site when the target nucleic acid is a wild type allele (iii) a blocking group linked at or near to the 3′ end of the primer; and   c) identifying resulting products based upon their mass.   
     
     
         121 . The method of  claim 120 , wherein the RNase H2 enzyme is thermostable. 
     
     
         122 . The method of  claim 120 , wherein the cleavage domain contains at least one ribonucleotide base. 
     
     
         123 . The method of  claim 120 , wherein two blocking groups are present at the 3′ end of the primer. 
     
     
         124 . A method for detecting a single base mutation in a target nucleic acid sample, the method comprising the steps of:
 a) mixing a sample containing the target nucleic acid, a primer extension reaction mixture, and an RNase H2 enzyme;   b) incubating the reaction mixture with at least one primer, the primer having a 5′ end and a 3′ end, said primer comprising (i) a 5′ sequence domain that is on the 5′ end of the region, said 5′ sequence domain being noncomplementary to the target nucleic acid sample and contains a secondary cleaving domain capable of being cleaved by RNase H2 when hybridized to a complementary base or sequence (ii) a region at least partially complementary to the target nucleic acid and is capable of supporting DNA replication, (iii) a cleaving domain that is capable of being cleaved by the RNase H2 enzyme when the primer hybridizes to the target nucleic acid and the cleaving domain is aligned and complementary to a potential base mutation site (iii) a blocking group linked at or near to the 3′ end of the primer; and   c) identifying resulting products based upon their mass.   
     
     
         125 . The method of  claim 124  wherein the blocking group is located internally from the 3′-terminal nucleotide. 
     
     
         126 . The method of  claim 125  wherein the blocking group is a C3 spacer. 
     
     
         127 . The method of  claim 125  further comprising at least a second blocking group at or near the 3′-end. 
     
     
         128 . The method of  claim 124 , wherein at least one RNA base in the cleaving domain contains one or more modified nucleotide residues selected from a group consisting of 2′ fluoro, 2′ alkyl, 2′ methyl, 2′ amino, 2′ LNA, 2′ ENA, 2′ thio, 2′-O-alkyl, 2′-O-methyl, 5′ thio, 5′ amine, 5′ alkyl, 5′ methylene, 5′ ethylene, 3′-phosphate and 3′-phosphate diester. 
     
     
         129 . The method of  claim 124 , wherein the reaction mixture contains on or more divalent cation of MgCl 2 , MnCl 2 , CoCl 2  or NiCl 2 . 
     
     
         130 . The method of  claim 124 , wherein the reaction mixture contains MgCl 2  and MnCl 2 . 
     
     
         131 . The method of  claim 124 , wherein the reaction mixture contains 3 mM MgCl 2  and 600 μM MnCl 2 . 
     
     
         132 . The method of  claim 124 , wherein the RNase H2 enzyme is from the group comprising  Pyrococcus abyssi, Sulfolobus solfataricus, Methanocaldococcus jannaschii, Pyrococcus kodakaraensis  and  Pyrococcus furiosus.    
     
     
         133 . An isolated thermostable ribonuclease H enzyme comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5. 
     
     
         134 . A method of sequencing a target nucleic acid comprising the steps of:
 a) providing a reaction mixture comprising the target nucleic acid, and acceptor oligonucleotide, a donor oligonucleotide set, the donor oligonucleotide set comprising four oligonucleotide groups corresponding to guanine, cytosine, adenine and thymine nucleotides, wherein each oligonucleotide group comprises donor oligonucleotides that are about 7 to 11 bases long containing a hairpin, wherein a first base domain on the 5′ end of the donor oligonucleotide is one to two bases and correspond to the nucleotide or nucleotides of the given set, a second base domain on the 5′ end of the donor oligonucleotide is at least one RNA residue or a modified residue that is a substrate for RNase H2, and remaining bases are degenerate or universal, and a label that is on the 3′ side of the RNA or modified residue, said label corresponding to the first base domain, and a blocking group at the 3′ end of the acceptor oligonucleotide;   b) hybridizing the target nucleic acid to the acceptor oligonucleotide and the donor oligonucleotide that is complementary to the target nucleic acid;   c) introducing a ligase enzyme to ligate the donor oligonucleotide and the acceptor oligonucleotide;   d) introducing an RNase H2 enzyme to cleave the donor oligonucleotide 5′ of the RNA or modified residue and leaving the first base domain on the 5′ end of the donor molecule attached to the acceptor oligonucleotide; and   e) repeating steps b-d.   
     
     
         135 . A method of synthesizing a target nucleic acid, comprising the steps of:
 a) providing a reaction mixture comprising an acceptor oligonucleotide, a donor oligonucleotide set, the donor oligonucleotide set comprising four oligonucleotide groups corresponding to guanine, cytosine, adenine and thymine nucleotides, wherein each oligonucleotide group comprises donor oligonucleotides containing a hairpin and an overhang on the 5′ end about 1 to 5 bases long, wherein the first base on the 5′ end of the donor oligonucleotide corresponds to the nucleotide of the given set, the second base on the 5′ end of the donor oligonucleotide is an RNA residue or a modified residue that is a substrate for RNase H2, remaining bases that are random or universal, and optionally a label that is on the 3′ side of the RNA or modified residue, said label corresponding to the first base, and a blocking group at the 3′ end of the acceptor oligonucleotide;   b) hybridizing the acceptor oligonucleotide and the donor oligonucleotide that is complementary to the acceptor oligonucleotide;   c) introducing a ligase enzyme to ligate the donor oligonucleotide and the acceptor oligonucleotide;   d) introducing an RNase H2 enzyme to cleave the donor oligonucleotide 5′ of the RNA or modified residue and leaving the first base on the 5′ end of the donor molecule attached to the acceptor oligonucleotide; and   e) repeating steps b-d.   
     
     
         136 . The method of  claim 135 , wherein a dideoxytriphosphate and a polymerase are added after the ligase enzyme and before the RNase H2 enzyme. 
     
     
         137 . The method of  claim 136 , wherein the polymerase is a terminal transferase. 
     
     
         138 . A method for sequencing a target nucleic acid comprising:
 a) mixing the target nucleic acid and a sequencing reaction mixture;   b) and incubating the reaction mixture with at least one primer of  claim 1  under suitable conditions.   
     
     
         139 . A method for detecting a single base mutation comprising the steps of:
 a) mixing a sample containing a nucleic acid and a PCR reaction mixture;   b) and incubating the reaction mixture with at least one primer of  claim 1  under suitable conditions.   
     
     
         140 . A method for ligating target nucleic acid segments comprising the steps of:
 a) adding an effective amount of at least one of the primers of  claim 1  to a ligation mixture comprising the target nucleic acid segments, a suitable buffer and a suitable ligase, and   b) incubating said mixture under suitable ligation conditions.