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 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.
2 . The DNA polymerase inhibitor of claim 1 , wherein the quencher comprises at least two different quenchers.
3 . The DNA polymerase inhibitor of claim 1 , further comprising a minor groove binder.
4 . The DNA polymerase inhibitor of claim 1 , 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.
5 . The DNA polymerase inhibitor of claim 4 , further comprising a minor groove binder.
6 . The DNA polymerase inhibitor of claim 5 , 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.
7 . 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.
8 . The method of claim 7 , wherein the target nucleic acid comprises RNA.
9 . The method of claim 7 , wherein the target nucleic acid comprises DNA.
10 . The method of claim 7 , wherein the primer comprises a primer pair and the at least one cycle of amplification comprises PCR.
11 . The method of claim 7 , 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.
12 . The method of claim 7 , 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.
13 . The method of claim 12 , wherein the nucleotide sequence of the DNA polymerase inhibitor is not extendible by the DNA polymerase.
14 . The method of claim 7 , wherein the DNA polymerase inhibitor further comprises a minor groove binder.
15 . The method of claim 7 , 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.
16 . The method of claim 15 , 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.
17 . The method of claim 7 , wherein the nucleotide sequence of the DNA polymerase inhibitor comprises an aptamer.
18 . The method of claim 7 , wherein the DNA polymerase inhibitor comprise sat least two different quenchers.
19 . The method of claim 7 , wherein the DNA polymerase inhibitor comprises a nucleotide analog.
20 . The method of claim 19 , wherein the nucleotide analog comprises a deaza-dA, a deaza-dG, a ddN, a LNA, a PNA, or combinations thereof.