Reagents and methods of pcr

Abstract

Modified double-stranded oligonucleotides that have terminal regions on each of their strands, that have a hybrid length of 6-50 nucleotides long, that have a melting temperature Tm of at least 32° C., and that include 2-4 modifying groups, each covalently attached to a different terminal region, preferably to a terminal nucleotide, said modifying groups being polycyclic substituents that do not have bulky portions that are non-planar, said modified oligonucleotide being capable of binding to the 5′ exonuclease domains of DNA polymerases and, when included in a PCR or other primer-dependent DNA amplification reaction at a concentration, generally not more than 2000 nM, that is effective for at least one of the functions of suppressing mispriming, increasing polymerase selectivity against 3′ terminal mismatches. increasing polymerase selectivity against AT-rich 3′ ends, reducing scatter among replicates, suppressing polymerase 5′ exonuclease activity, and inhibiting polymerase activity; as well as amplification reaction mixtures containing such modified double-stranded oligonucleotides, and amplification reactions, amplification assays and kits that include such modified double-stranded oligonucleotides.

What is claimed is: 
     
         1 . In a reaction mixture for a primer-dependent DNA amplification reaction including primer extension by a DNA polymerase for amplifying at least one DNA target sequence, said reaction mixture including at least one primer pair, a DNA polymerase and dNTP's, the improvement comprising including in the reaction mixture prior to the start of amplification at least one double-stranded oligonucleotide additive that has a hybrid length of 6-50 nucleotides long, that is at least fifty percent double-stranded at 32° C., that has terminal regions on each of its strands and includes 1-4 modifying groups, each covalently attached to a different terminal region, said modifying groups being polycyclic moieties that do not have bulky portions that are non-planar, wherein said at least one double-stranded oligonucleotide additive is included at a concentration relative to the concentration of said DNA polymeras that is effective for at least one of the functions of suppressing mispriming, increasing polymerase selectivity against hybrids having recessed 3′ terminal sequences that are not perfectly complementary, increasing polymerase selectivity against hybrids having recessed 3′ terminal sequences that are AT-rich, reducing scatter among replicate reactions, inhibiting polymerase 5′ exonuclease activity, and inhibiting polymerase activity; provided that, if the additive is a primer or detection probe for any target sequence, it includes at least three modifying groups. 
     
     
         2 . The amplification reaction mixture of  claim 1  wherein the at least one modifying groups is 2-4 modifying groups. 
     
     
         3 . The amplification reaction mixture of  claim 2  wherein the 2-4 modifying groups is three modifying groups. 
     
     
         4 . The amplification reaction mixture of  claim 3  wherein the additive includes a first strand that is a primer or probe for said at least one target sequence and a reverse complement strand that is partially complementary to the first strand. 
     
     
         5 . The amplification reaction mixture of  claim 2  wherein the 2-4 modifying groups is four modifying groups. 
     
     
         6 . The amplification reaction mixture of any of  claims 1 - 5  wherein the modifying groups are covalently linked to terminal nucleotides of said at least one double-stranded oligonucleotide additive. 
     
     
         7 . The amplification reaction mixture of any of  claims 1 - 6  wherein the modifying groups are Dabcyl. 
     
     
         8 . The amplification reaction mixture of  claim 2  wherein the at least one additive is a mixture of two additives. 
     
     
         9 . The amplification reaction mixture of  claim 8  wherein the mixture consists of three strands. 
     
     
         10 . The amplification reaction mixture of any of  claims 1 - 9 , wherein the at least one double-stranded oligonucleotide additive consists of natural nucleotides. 
     
     
         11 . The amplification reaction mixture of any of  claims 1 - 9  wherein the at least one double-stranded oligonucleotide additive is DNA. 
     
     
         12 . The amplification reaction mixture according to any of  claims 1 - 3  and  5 - 11  wherein the at least one additive is not a primer or probe for said at least one target sequence. 
     
     
         13 . The amplification reaction mixture according to any of  claims 1 - 12  wherein the concentration of said at least one double-stranded oligonucleotide additive is not more than 1000 nM. 
     
     
         14 . The amplification reaction mixture according to any of  claims 1 - 13 , further including a reverse transcriptase. 
     
     
         15 . The amplification reaction mixture of  claim 1  wherein the at least one double-stranded additive includes from one to four single-stranded overhangs. 
     
     
         16 . The amplification reaction mixture of  claim 15  wherein the at least one double-stranded additive comprises at least one strand that, when not hybridized, forms a stem-loop structure. 
     
     
         17 . A method for amplifying at least one DNA target sequence comprising contacting said at least and DNA target sequence with an amplification reaction mixture according to  claim 1  and subjecting the reaction mixture to a primer-dependent DNA amplification reaction having a primer annealing temperature and a primer extension temperature. 
     
     
         18 . The method of  claim 17  wherein contacting the at least one DNA target sequence with the reaction mixture consists of adding the at least one DNA target sequence in single-stranded form to the reaction mixture. 
     
     
         19 . The method of  claim 17  that includes reverse transcribing RNA to obtain the at least one DNA target sequence. 
     
     
         20 . The method of  claim 17  wherein the at least one modifier is 2-4 modifiers. 
     
     
         21 . The method of  claim 20  wherein the double-stranded oligonucleotide has a melting temperature, Tm, that is in the range of the primer annealing temperature to not more than 5° C. below the primer annealing temperature. 
     
     
         22 . The method of  claim 20  wherein the double-stranded oligonucleotide has a melting temperature, Tm, that is higher than the primer annealing temperature. 
     
     
         23 . The method of  claim 17  wherein the additive has three modifying groups and includes a first strand that is a primer or probe for said at least one target sequence and a reverse complement strand that is partially complementary to the first strand. 
     
     
         24 . The method of  claim 20  wherein said at least one additive is a mixture of two additives. 
     
     
         25 . The method of  claim 22  wherein the mixture includes a first additive having a double-stranded oligonucleotide that has a Tm that is in the range of the primer annealing temperature to not more than 5° C. below the primer annealing temperature and a second additive having a double-stranded oligonucleotide that has a Tm that is higher than the primer annealing temperature. 
     
     
         26 . The method of  claim 25  wherein each additive includes 3-4 modifying groups. 
     
     
         27 . The method according to any of  claims 17 - 26 , wherein the primer-dependent amplification reaction is a polymerase chain reaction (PCR) amplification reaction. 
     
     
         28 . The method according to any of  claims 17 - 26 , wherein the primer-dependent amplification is a LATE-PCR amplification reaction. 
     
     
         29 . The method according to any of  claims 17 - 28  wherein the at least one DNA target sequence is reverse transcribed from an RNA target sequence. 
     
     
         30 . An amplification assay that includes amplification according to any of  claims 17 - 29  and fluorescence detection of single-stranded products of the reaction, double-stranded products of the reaction, or both, either in real time during amplification or end point following amplification, wherein double-stranded products of the reaction are detected with a fluorescent DNA dye, single-stranded products of the reaction are detected with at least one fluorescently labeled hybridization probe, or both. 
     
     
         31 . A kit of reagents amplifying at least one DNA target sequence, said kit including the reagents for a reaction mixture according to any of  claims 1 - 16 . 
     
     
         32 . The kit according  claim 31  further including reverse transcriptase. 
     
     
         33 . The kit according to  claim 31  or  claim 32  further including a DNA dye. 
     
     
         34 . The kit according to any of  claims 31 - 33  further including a fluorescently labeled detection probe for said at least one target sequence. 
     
     
         35 . A modified double-stranded oligonucleotide that has terminal regions on each of its strands, that has a hybrid length of 6-50 nucleotides long, that is at least fifty percent double-stranded at 32° C., and that includes 2-4 modifying groups, each covalently attached to a different terminal region, said modifying groups being polycyclic moieties that do not have bulky portions that are non-planar, said modified oligonucleotide being capable of inhibiting the 5′ exonuclease domains of DNA polymerases. 
     
     
         36 . The modified oligonucleotide of  claim 35  wherein the double-stranded oligonucleotide is DNA. 
     
     
         37 . The modified oligonucleotide of  claim 35  or  claim 36  wherein said modifying groups are attached to terminal nucleotides. 
     
     
         38 . A mixture of two modified double-stranded oligonucleotides according to any of  claims 35 - 37 . 
     
     
         39 . The mixture of  claim 38  wherein the two double-stranded oligonucleotides comprise three strands. 
     
     
         40 . The amplification reaction mixture of  claim 1 , wherein the DNA polymerase is thermostable.