Compositions, methods, and kits for amplifying nucleic acids

Abstract

The present teachings are directed to compositions, methods, and kits for amplifying target nucleic acids while reducing non-specific fluorescence and undesired amplification products, sometimes referred to as secondary amplification products or spurious side-products. The enzyme inhibitors disclosed herein comprise a nucleotide sequence and at least one quencher. Complexes comprising an enzyme inhibitor associated with an enzyme, wherein at least one enzymatic activity of the enzyme is inhibited, are also provided. Methods for amplifying a target nucleic acid while reducing undesired amplification products are disclosed, as are methods for reducing non-specific fluorescence. Kits for expediting the performance of certain disclosed methods are also provided.

1 . A complex comprising a DNA polymerase and a DNA polymerase inhibitor, wherein the DNA polymerase inhibitor comprises a nucleotide sequence and a quencher.  
     
     
         2 . The complex of  claim 1 , further comprising a nucleotide triphosphate (NTP), a nucleotide analog, or a NTP and a nucleotide analog.  
     
     
         3 . The complex of  claim 1 , wherein the nucleotide sequence of the DNA polymerase inhibitor comprises a first region, a second region, a third region, and optionally, a fourth region; and wherein the first region is complementary to the third region.  
     
     
         4 . The complex of  claim 3 , further comprising a NTP, a nucleotide analog, or a NTP and a nucleotide analog.  
     
     
         5 . The complex of  claim 3 , wherein the nucleotide sequence is not extendible by the DNA polymerase.  
     
     
         6 . The complex of  claim 3 , wherein the DNA polymerase inhibitor further comprises a minor groove binder.  
     
     
         7 . The complex of  claim 1 , wherein the nucleotide sequence comprises a first oligonucleotide and a second oligonucleotide, wherein the first oligonucleotide comprises a first region and the second oligonucleotide comprises a third region and optionally, a fourth region, and wherein the first region of the first oligonucleotide is complementary to the third region of the second oligonucleotide.  
     
     
         8 . The complex of  claim 7 , further comprising a NTP, a nucleotide analog, or a NTP and a nucleotide analog.  
     
     
         9 . The complex of  claim 7 , wherein the first oligonucleotide is not extendible by the DNA polymerase, the second oligonucleotide is not extendible by the DNA polymerase, or the first oligonucleotide and the second oligonucleotide are not extendible by the DNA polymerase.  
     
     
         10 . The complex of  claim 1 , wherein the nucleotide sequence of the DNA polymerase inhibitor comprises an aptamer.  
     
     
         11 . The complex of  claim 1 , wherein the DNA polymerase inhibitor comprises at least two different quenchers.  
     
     
         12 . The complex of  claim 1 , wherein the nucleotide sequence of the DNA polymerase inhibitor comprises a nucleotide analog.  
     
     
         13 . The complex of  claim 12 , wherein the nucleotide analog comprises a 7-deaza-2′-deoxyadenosine (deaza-dA), a 7-deaza-2′-deoxyguanosine (deaza-dG), a dideoxynucleotide (ddN), a locked nucleic acid (LNA), a peptide nucleic acid (PNA), or combinations thereof.  
     
     
         14 . A complex comprising a DNA polymerase, a DNA polymerase inhibitor, and optionally a NTP, a nucleotide analog, or a NTP and a nucleotide analog, wherein the DNA polymerase inhibitor comprises a nucleotide sequence and a quencher; wherein the nucleotide sequence comprises a first region, a second region, a third region, and optionally, a fourth region; wherein the first region is complementary to the third region; wherein the first region, the third region, or the first region and the third region comprise at least one nucleotide analog; and wherein the first region comprises a first quencher and the second region comprises a second quencher.  
     
     
         15 . The complex of  claim 14 , wherein the third region is not extendible by the DNA polymerase or the fourth region is not extendible by the DNA polymerase.  
     
     
         16 . The complex of  claim 14 , wherein the nucleotide analog comprises a deaza-dA, a deaza-dG, a ddN, a LNA, a PNA, or combinations thereof.  
     
     
         17 . The complex of  claim 14 , wherein the DNA polymerase inhibitor further comprises a minor groove binder.  
     
     
         18 . A DNA polymerase inhibitor comprising a nucleotide sequence and a quencher.  
     
     
         19 . The DNA polymerase inhibitor of  claim 18 , wherein the nucleotide sequence of the DNA polymerase inhibitor comprises a first region, a second region, a third region, and optionally, a fourth region; and wherein the first region is complementary to the third region.  
     
     
         20 . The DNA polymerase inhibitor of  claim 19 , wherein the nucleotide sequence is not extendible by a DNA polymerase.  
     
     
         21 . The DNA polymerase inhibitor of  claim 19 , further comprising a minor groove binder.  
     
     
         22 . The DNA polymerase inhibitor of  claim 18  wherein the nucleotide sequence comprises a first oligonucleotide and a second oligonucleotide, wherein the first oligonucleotide comprises a first region and the second oligonucleotide comprises a third region and optionally, a fourth region, and wherein the first region of the first oligonucleotide is complementary to the third region of the second oligonucleotide.  
     
     
         23 . The DNA polymerase inhibitor of  claim 22 , wherein the first oligonucleotide is not extendible by a DNA polymerase, the second oligonucleotide is not extendible by a DNA polymerase, or the first oligonucleotide and the second oligonucleotide are not extendible by a DNA polymerase.  
     
     
         24 . The DNA polymerase inhibitor of  claim 18 , wherein the nucleotide sequence of the DNA polymerase inhibitor comprises an aptamer.  
     
     
         25 . The DNA polymerase inhibitor of  claim 18 , wherein the DNA polymerase inhibitor comprises at least two different quenchers.  
     
     
         26 . The DNA polymerase inhibitor of  claim 18 , further comprising a nucleotide analog.  
     
     
         27 . The DNA polymerase inhibitor of  claim 26 , wherein the nucleotide analog comprises a deaza-dA, a deaza-dG, a ddN, a LNA, a PNA, or combinations thereof.  
     
     
         28 . A DNA polymerase inhibitor comprising a nucleotide sequence and a quencher, wherein the nucleotide sequence comprises a first region, a second region, a third region, and optionally, a fourth region; wherein the first region is complementary to the third region; wherein the first region comprises a nucleotide analog, the third region comprises a nucleotide analog, or the first region and the third region comprise a nucleotide analog; and wherein the first region comprises a first quencher and the second region comprises a second quencher.  
     
     
         29 . The DNA polymerase inhibitor of  claim 28 , wherein the nucleotide sequence is not extendible by a DNA polymerase.  
     
     
         30 . The DNA polymerase inhibitor of  claim 28 , further comprising a minor groove binder.  
     
     
         31 . The DNA polymerase inhibitor of  claim 28 , wherein the nucleotide analog comprises a deaza-dA, a deaza-dG, a ddN, a PNA, a LNA, or combinations thereof.  
     
     
         32 . A DNA polymerase inhibitor comprising a nucleotide sequence and a quencher, wherein the nucleotide sequence comprises 5′-TCTGGGATA(deaza-dA)TT(deaza-dA)TGGTA(deaza-dA)ATATG (Tn )C(deaza-dA)TATTTATT(deaza-dA)TA(deaza-dA)TTATC-3′, and wherein Tn comprises TT, TTT, TTTT, TTTTT, or TTTTTT.  
     
     
         33 . The DNA polymerase inhibitor of  claim 32 , wherein the quencher comprises at least two different quenchers.  
     
     
         34 . The DNA polymerase inhibitor of  claim 32 , further comprising a minor groove binder.  
     
     
         35 . The DNA polymerase inhibitor of  claim 32 , wherein the nucleotide sequence comprises: 5′-TCTGGGATA(deaza-dA)TT(deaza-dA)TGGTA(deaza-dA)ATATGTTTTC(deaza-dA)TATTTATT(deaza-dA)TA(deaza-dA)TTATC-3′, and the quencher comprises at least two different quenchers.  
     
     
         36 . The DNA polymerase inhibitor of  claim 35 , further comprising a minor groove binder.  
     
     
         37 . The DNA polymerase inhibitor of  claim 36 , wherein: the first quencher comprises at least one of DABCYL, DABSYL, TAMRA, TET, and ROX; and the minor groove binder further comprises the second quencher.  
     
     
         38 . A method for reducing non-specific fluorescence comprising: 
 forming a reaction composition comprising a DNA polymerase, a DNA polymerase inhibitor comprising a nucleotide sequence and a quencher, a nucleotide triphosphate (NTP), a target nucleic acid, a primer, a nucleic acid dye, and optionally a nucleotide analog, at a first temperature, wherein the nucleotide sequence comprises at least one double-stranded segment, wherein the DNA polymerase and the DNA polymerase inhibitor associate to form a complex, and wherein the quencher inhibits fluorescence of the nucleic acid dye associated with the double-stranded segment of the nucleotide sequence;    heating the reaction composition to a second temperature to dissociate the complex;    subjecting the reaction composition to at least one cycle of amplification to generate a multiplicity of amplicons; and    detecting the fluorescence of the nucleic acid dye associated with the multiplicity of amplicons in the reaction composition, wherein the quencher inhibits fluorescence of the nucleic acid dye associated with the double-stranded segment of the nucleotide sequence of the DNA polymerase inhibitor.    
     
     
         39 . The method of  claim 38 , wherein the detecting comprises real time detection.  
     
     
         40 . The method of  claim 38 , wherein the detecting comprises end-point detection.  
     
     
         41 . The method of  claim 40 , wherein the end-point detection comprises a melting curve analysis.  
     
     
         42 . The method of  claim 38 , wherein the DNA polymerase,the DNA polymerase inhibitor, and optionally a NTP and/or a nucleotide analog are incubated together to form a complex prior to the forming the reaction composition at the first temperature.  
     
     
         43 . The method of  claim 38 , wherein the nucleotide sequence of the DNA polymerase inhibitor comprises a first region, a second region, a third region, and optionally, a fourth region; and wherein the first region is complementary to the third region.  
     
     
         44 . The method of  claim 43 , wherein the nucleotide sequence is not extendible by the DNA polymerase.  
     
     
         45 . The method of  claim 38 , wherein the DNA polymerase inhibitor further comprises a minor groove binder.  
     
     
         46 . The method of  claim 38 , wherein the nucleotide sequence of the DNA polymerase inhibitor comprises a first oligonucleotide and a second oligonucleotide, wherein the first oligonucleotide comprises a first region and the second oligonucleotide comprises a third region and optionally, a fourth region, and wherein the first region of the first oligonucleotide is complementary to the third region of the second oligonucleotide.  
     
     
         47 . The method of  claim 46 , wherein the first oligonucleotide is not extendible by the DNA polymerase, the second oligonucleotide is not extendible by the DNA polymerase, or the first oligonucleotide and the second oligonucleotide are not extendible by the DNA polymerase.  
     
     
         48 . The method of  claim 38 , wherein the nucleotide sequence of the DNA polymerase inhibitor comprises an aptamer.  
     
     
         49 . The method of  claim 38 , wherein the DNA polymerase inhibitor comprises at least two different quenchers.  
     
     
         50 . The method of  claim 38 , wherein the DNA polymerase inhibitor comprises a nucleotide analog.  
     
     
         51 . The method of  claim 50 , wherein the nucleotide analog comprises a deaza-dA, a deaza-dG, a ddN, a LNA, a PNA, or combinations thereof.  
     
     
         52 . The method of  claim 38 , wherein the target nucleic acid comprises a multiplicity of different target nucleic acids, the primer comprises a multiplicity of different primers, and the multiplicity of amplicons comprises a multiplicity of different amplicons.  
     
     
         53 . The method of  claim 38 , wherein the detecting further comprises a detector probe.  
     
     
         54 . The method of  claim 38 , wherein the target nucleic acid comprises RNA.  
     
     
         55 . The method of  claim 54 , wherein the RNA comprises messenger RNA (mRNA).  
     
     
         56 . The method of  claim 54 , wherein the RNA comprises a small RNA molecule.  
     
     
         57 . The method of  claim 56 , wherein the small RNA molecule comprises a microRNA (miRNA).  
     
     
         58 . The method of  claim 38 , wherein the first temperature is about 22° C. to about 40° C.  
     
     
         59 . The method of  claim 38 , wherein the second temperature is about 48° C. to about 73° C.  
     
     
         60 . The method of  claim 59 , wherein the second temperature is about 53° C. to about 67° C.  
     
     
         61 . The method of  claim 60 , wherein the second temperature is 64° C. to 67° C.  
     
     
         62 . The method of  claim 38 , wherein the target nucleic acid comprises DNA.  
     
     
         63 . The method of  claim 38 , wherein the primer comprises a primer pair and the at least one cycle of amplification comprises the DNA polymerase chain reaction (PCR).  
     
     
         64 . A method for amplifying a target nucleic acid comprising: 
 forming a reaction composition comprising a DNA polymerase, a DNA polymerase inhibitor comprising a nucleotide sequence and a quencher, a NTP, the target nucleic acid, a primer, a nucleic acid dye, and optionally a nucleotide analog, at a first temperature, wherein the nucleotide sequence comprises at least one double-stranded segment, wherein the DNA polymerase and the DNA polymerase inhibitor associate to form a complex, and wherein the quencher inhibits fluorescence associated with the double-stranded segment of the nucleotide sequence;    heating the reaction composition to a second temperature to dissociate the complex; and    subjecting the reaction composition to at least one cycle of amplification to generate a multiplicity of amplicons.    
     
     
         65 . The method of  claim 64 , wherein the target nucleic acid comprises RNA.  
     
     
         66 . The method of  claim 65 , wherein the RNA comprises mRNA.  
     
     
         67 . The method of  claim 65 , wherein the RNA comprises a small RNA molecule.  
     
     
         68 . The method of  claim 67 , wherein the small RNA molecule comprises a miRNA.  
     
     
         69 . The method of  claim 64 , wherein the target nucleic acid comprises DNA.  
     
     
         70 . The method of  claim 64 , wherein the primer comprises a primer pair and the at least one cycle of amplification comprises PCR.  
     
     
         71 . The method of  claim 64 , wherein the DNA polymerase the DNA polymerase inhibitor, and optionally a NTP and/or a nucleotide analog, are incubated together to form a complex prior to the forming the reaction composition at the first temperature.  
     
     
         72 . The method of  claim 64 , wherein the nucleotide sequence of the DNA polymerase inhibitor comprises a first region, a second region, a third region, and optionally, a fourth region; and wherein the first region is complementary to the third region.  
     
     
         73 . The method of  claim 72 , wherein the nucleotide sequence of the DNA polymerase inhibitor is not extendible by the DNA polymerase.  
     
     
         74 . The method of  claim 64 , wherein the DNA polymerase inhibitor further comprises a minor groove binder.  
     
     
         75 . The method of  claim 64 , wherein the nucleotide sequence of the DNA polymerase inhibitor comprises a first oligonucleotide and a second oligonucleotide, wherein the first oligonucleotide comprises a first region and the second oligonucleotide comprises a third region and optionally, a fourth region, and wherein the first region of the first oligonucleotide is complementary to the third region of the second oligonucleotide.  
     
     
         76 . The method of  claim 75 , wherein the first oligonucleotide is not extendible by the DNA polymerase, the second oligonucleotide is not extendible by the DNA polymerase, or the first oligonucleotide and the second oligonucleotide are not extendible by the DNA polymerase.  
     
     
         77 . The method of  claim 64 , wherein the nucleotide sequence of the DNA polymerase inhibitor comprises an aptamer.  
     
     
         78 . The method of  claim 64 , wherein the DNA polymerase inhibitor comprises at least two different quenchers.  
     
     
         79 . The method of  claim 64 , wherein the DNA polymerase inhibitor comprises a nucleotide analog.  
     
     
         80 . The method of  claim 79 , wherein the nucleotide analog comprises a deaza-dA, a deaza-dG, a ddN, a LNA, a PNA, or combinations thereof.  
     
     
         81 . The method of  claim 64 , wherein: the target nucleic acid comprises a multiplicity of different target nucleic acids; the primer comprises a multiplicity of different primers, a multiplicity of different primer pairs, or combinations thereof; and the multiplicity of amplicons comprises a multiplicity of different amplicons.  
     
     
         82 . The method of  claim 64 , wherein the first temperature is about 22° C. to about 40° C.  
     
     
         83 . The method of  claim 64 , wherein the second temperature is about 48° C. to about 73° C.  
     
     
         84 . The method of  claim 83 , wherein the second temperature is about 53° C. to about 67° C.  
     
     
         85 . The method of  claim 83  wherein the second temperature is 63° C. to 67° C.  
     
     
         86 . A kit comprising a DNA polymerase inhibitor comprising a nucleotide sequence and a quencher.  
     
     
         87 . The kit of  claim 86 , wherein the nucleotide sequence of the DNA polymerase inhibitor comprises an aptamer.  
     
     
         88 . The kit of  claim 86 , wherein the nucleotide sequence comprises a first region, a second region, a third region, and optionally, a fourth region; wherein the first region is complementary to the third region.  
     
     
         89 . The kit of  claim 88 , wherein the first region, the third region, or the first region and the third region comprise a nucleotide analog; and wherein the first region comprises a first quencher, the second region comprises a second quencher, or the first region comprises a first quencher and the second region comprises a second quencher.  
     
     
         90 . The kit of  claim 86 , wherein the nucleotide sequence comprises a first oligonucleotide and a second oligonucleotide, wherein the first oligonucleotide comprises a first region and the second oligonucleotide comprises a third region and optionally, a fourth region, and wherein the first region of the first oligonucleotide is complementary to the third region of the second oligonucleotide.  
     
     
         91 . The kit of  claim 86 , wherein the DNA polymerase inhibitor further comprises a minor groove binder.  
     
     
         92 . The kit of  claim 86 , further comprising a nucleic acid dye.  
     
     
         93 . The kit of  claim 92 , wherein the nucleic acid dye comprises, ethidium bromide, 4′,6-diamidino-2-phenylindole (DAPI), an unsymmetrical cyanine dye, or combinations thereof.  
     
     
         94 . The kit of  claim 93 , wherein the unsymmetrical cyanine dye comprises [2-[N-(3-dimethylaminopropyl)-N-propylamino]-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenyl-quinolinium] (SYBR® Green), [2-[N-bis-(3-dimethylaminopropyl)-amino)-amino]-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenyl-quinolinium] (PicoGreen®), 4-[(3-methyl-6-(benzothiazol-2-yl )-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidene)-1-methyl-pyridinium iodide (BEBO), BOXTO, BETO, or combinations thereof.  
     
     
         95 . The kit of  claim 86 , wherein the nucleotide sequence of the DNA polymerase inhibitor comprises an aptamer.  
     
     
         96 . The kit of  claim 86 , wherein the nucleotide sequence comprises a first region, a second region, a third region, and optionally, a fourth region; wherein the first region is complementary to the third region.  
     
     
         97 . The kit of  claim 86 , further comprising a reporter probe.  
     
     
         98 . A complex comprising an enzyme and an enzyme inhibitor, wherein the enzyme inhibitor comprises a nucleotide sequence and a quencher.  
     
     
         99 . The complex of  claim 98 , wherein the enzyme comprises an RNA polymerase, a ligase, a helicase, a cleaving enzyme, or combinations thereof.  
     
     
         100 . The complex of  claim 98 , wherein the nucleotide sequence of the enzyme inhibitor comprises an aptamer.  
     
     
         101 . The complex of  claim 98 , wherein the nucleotide sequence of the inhibitor comprises a first region, a second region, a third region, and optionally, a fourth region; and wherein the first region is complementary to the third region.  
     
     
         102 . The complex of  claim 98 , wherein the nucleotide sequence of the enzyme inhibitor comprises one oligonucleotide, two oligonucleotides, or three oligonucleotides.  
     
     
         103 . The complex of  claim 98 , wherein the enzyme inhibitor comprises at least two quenchers.  
     
     
         104 . An enzyme inhibitor comprising a nucleotide sequence and a quencher.  
     
     
         105 . The enzyme inhibitor of  claim 103 , wherein the nucleotide sequence comprises an aptamer.  
     
     
         106 . The enzyme inhibitor of  claim 104 , wherein the nucleotide sequence of the enzyme inhibitor comprises one oligonucleotide, two oligonucleotides, or three oligonucleotides.  
     
     
         107 . The enzyme inhibitor of  claim 104 , wherein the nucleotide sequence comprises a first region, a second region, a third region, a fourth region, a fifth region, and a sixth region; wherein the first region is complementary to the third region and the fourth region is complementary with the sixth region.  
     
     
         108 . The enzyme inhibitor of  claim 107 , further comprising a non-ligatable nucleotide.  
     
     
         109 . The enzyme inhibitor of  claim 107 , further comprising a non-cleavable flap sequence.  
     
     
         110 . The enzyme inhibitor of  claim 109 , wherein the non-cleavable flap sequence comprises an uncleavable internucleotide linkage.  
     
     
         111 . A method for reducing non-specific fluorescence comprising: 
 forming a reaction composition comprising an enzyme, an enzyme inhibitor comprising a nucleotide sequence and a quencher, a target nucleic acid, a primer, and a nucleic acid dye, at a first temperature, wherein the nucleotide sequence can form at least one double-stranded segment, wherein the enzyme and the enzyme inhibitor associate to form a complex, and wherein the quencher inhibits fluorescence of the nucleic acid dye associated with the double-stranded segment of the nucleotide sequence of the enzyme inhibitor;    heating the reaction composition to a second temperature to dissociate the complex;    amplifying the target nucleic acid in the reaction composition to generate a multiplicity of amplicons; and    detecting the fluorescence of the nucleic acid dye associated with the multiplicity of amplicons in the reaction composition, wherein the quencher inhibits fluorescence of the nucleic acid dye associated with the double-stranded segment of the nucleotide sequence of the enzyme inhibitor.    
     
     
         112 . The method of  claim 111 , wherein the enzyme comprises an RNA polymerase, a ligase, a helicase, a cleaving enzyme, or combinations thereof.  
     
     
         113 . The method of  claim 111 , wherein the nucleotide sequence of the enzyme inhibitor comprises an aptamer.  
     
     
         114 . The method of  claim 111 , wherein the nucleotide sequence of the enzyme inhibitor comprises one oligonucleotide, two oligonucleotides, or three oligonucleotides.  
     
     
         115 . The method of  claim 111 , wherein the reaction composition comprises a primer, a primer pair, a ligation probe pair, a cleavage probe pair, or combinations thereof.  
     
     
         116 . A method for amplifying a target nucleic acid comprising: 
 forming a reaction composition comprising an enzyme, an enzyme inhibitor, the target nucleic acid, and a nucleic acid dye, at a first temperature; wherein the enzyme inhibitor comprises a nucleotide sequence and at least one quencher; wherein the nucleotide sequence can form at least one double-stranded segment; wherein the enzyme and the enzyme inhibitor associate to form an enzyme-enzyme inhibitor complex; and wherein the at least one quencher inhibits fluorescence associated with the double-stranded segment of the nucleotide sequence;    heating the reaction composition to a second temperature to dissociate the complex; and    amplifying the target nucleic acid in the reaction composition to generate a multiplicity of amplicons.    
     
     
         117 . The method of  claim 116 , wherein the enzyme comprises an RNA polymerase, a ligase, a helicase, a cleaving enzyme, or combinations thereof.  
     
     
         118 . The method of  claim 116 , wherein the nucleotide sequence of the enzyme inhibitor comprises an aptamer.  
     
     
         119 . The method of  claim 116 , wherein the nucleotide sequence of the enzyme inhibitor comprises one oligonucleotide, two oligonucleotides, or three oligonucleotides.  
     
     
         120 . The method of  claim 116 , wherein the reaction composition comprises a primer, a primer pair, a ligation probe pair, a cleavage probe pair, or combinations thereof.  
     
     
         121 . A kit comprising an enzyme inhibitor that comprises a nucleotide sequence and a quencher.  
     
     
         122 . The kit of  claim 121 , wherein the enzyme inhibitor comprises an RNA polymerase inhibitor, a ligase inhibitor, a helicase inhibitor, a cleaving enzyme inhibitor, or combinations thereof.  
     
     
         123 . The kit of  claim 122 , further comprising a primer, a DNA polymerase, a ligase, or combinations thereof.