COMPOSITIONS, METHODS, AND KITS FOR AMPLIFYING NUCLEIC ACIDS
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 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.
2 . The method of claim 1 , wherein the enzyme comprises an RNA polymerase, a ligase, a helicase, a cleaving enzyme, or combinations thereof.
3 . The method of claim 1 , wherein the nucleotide sequence of the enzyme inhibitor comprises an aptamer.
4 . The method of claim 1 , wherein the nucleotide sequence of the enzyme inhibitor comprises one oligonucleotide, two oligonucleotides, or three oligonucleotides.
5 . The method of claim 1 , wherein the reaction composition comprises a primer, a primer pair, a ligation probe pair, a cleavage probe pair, or combinations thereof.
6 . 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.
7 . The method of claim 6 , wherein the enzyme comprises an RNA polymerase, a ligase, a helicase, a cleaving enzyme, or combinations thereof.
8 . The method of claim 6 , wherein the nucleotide sequence of the enzyme inhibitor comprises an aptamer.
9 . The method of claim 6 , wherein the nucleotide sequence of the enzyme inhibitor comprises one oligonucleotide, two oligonucleotides, or three oligonucleotides.
10 . The method of claim 6 , wherein the reaction composition comprises a primer, a primer pair, a ligation probe pair, a cleavage probe pair, or combinations thereof.