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 complex comprising a DNA polymerase and an inhibitor of the DNA polymerase, the inhibitor comprising a contiguous nucleotide sequence configured as a stem-loop structure, and at least a first and a second quencher, wherein the contiguous nucleotide sequence comprises a first region, a second region, a third region, and optionally, a fourth region; wherein the first region and the third region anneal to form a double-stranded stem segment; wherein the first region and/or the third region comprise at least one nucleotide analog; and wherein the first region, or the fourth region if present, comprises the first quencher and the second region forms a loop and comprises the second quencher, wherein the at least one nucleotide analog is selected from the group consisting of a deaza-dA, a deaza-dG, a ddN, or any combination thereof; wherein the first and/or the second quencher is/are selected from the group consisting of a DABCYL moiety, a DABSYL moiety, a TAMRA moiety, a TET moiety, and a ROX moiety; wherein the nucleotide sequence of the DNA polymerase inhibitor comprises an aptamer; and wherein the DNA polymerase is a Taq DNA polymerase.
2. The complex of claim 1 , wherein the inhibitor is not extendible by the DNA polymerase.
3. The complex of claim 1 , wherein the first and/or the second quencher is DABCYL.
4. The complex of claim 1 , wherein the at least one nucleotide analog is a dideoxynucleotide.
5. The complex of claim 4 , wherein the dideoxynucleotide is ddC.
6. The complex of claim 1 , wherein the DNA polymerase inhibitor further comprises a minor groove binder.
7. The complex of claim 1 , wherein the DNA polymerase inhibitor comprises 5′-TCTGGGATA(deaza-dA)TT(deaza-dA)TGGTA(deaza-dA)ATATG(Tn)C(deaza-dA)TATTTATT(deaza-dA)TA(deaza-dA)TTATC-3′, wherein the Tn is TT, TTT, TTTT, TTTTT, or TTTTTT.
8. The complex of claim 1 , wherein the first and the second quenchers are the same.
9. The complex of claim 1 , wherein the fourth region comprises a 5′ overhang.
10. The complex of claim 1 , wherein the first region comprises at least one mismatched base pair when annealed to the third region.
11. The complex of claim 10 , wherein the at least one mismatched base pair is a G:T base pair.