Polymerase inhibitor and method of using same
View Patent ↗The present invention provides nucleic acid based polymerase inhibitors and methods for reducing non-specific polymerase extension and amplification in nucleic acid amplification reactions. The polymerase inhibitors provide a double stranded nucleic acid portion that is recognized by a polymerase enzyme as a template for extension but is incapable of being extended by the polymerase enzyme. The polymerase binds to the polymerase inhibitor which sequesters the enzyme until the temperature achieves a level that denatures the double stranded portion of the inhibitor after which the polymerase is released and can then catalyze nucleic acid extension.
1. A polymerase inhibitor comprising a nucleic acid sequence a portion of which is double-stranded at or below the melting temperature of the nucleic acid sequence, wherein the double-stranded portion of the nucleic acid sequence is of sufficient length to be recognized by a polymerase as a template for extension except that the nucleic acid sequence is substantially incapable of being extended by the polymerase, wherein at least a 5′ nucleotide of the nucleic acid sequence is single stranded when the nucleic acid is at or below the melting temperature of the nucleic acid sequence.
2. The polymerase inhibitor of claim 1 wherein the polymerase inhibitor is not specific for a nucleic acid polymerase or family of polymerases related to the nucleic acid polymerase.
3. The polymerase inhibitor of claim 1 wherein the inhibitory activity of the polymerase inhibitor is not sequence specific.
4. The polymerase inhibitor of claim 1 wherein the nucleic acid sequence of the polymerase inhibitor does not act as a primer for an intended target nucleic acid.
5. The polymerase inhibitor of claim 1 wherein the nucleic acid sequence of the polymerase inhibitor does not form part of the nucleic acid that includes an intended target nucleic acid sequence.
6. The polymerase inhibitor claim 1 wherein the polymerase inhibitor consists essentially of a nucleic acid sequence.
7. The polymerase inhibitor of claim 1 wherein the 3′ terminal nucleic acid of the nucleic acid sequence comprises a blocking moiety that prevents extension of the 3′ terminal nucleic acid by the polymerase.
8. The polymerase inhibitor of claim 1 wherein the portion of the nucleic acid that is double-stranded at or below the melting temperature of the nucleic acid is formed by a single nucleic acid sequence that is capable of annealing to itself.
9. The polymerase inhibitor of claim 1 wherein the portion of the nucleic acid that is double-stranded at or below the melting temperature of the nucleic acid is formed by two separate nucleic acid sequences that at least partially anneal to one another.
10. The polymerase inhibitor of claim 1 wherein at least the 3′ terminal nucleic acid of the nucleic acid sequence is single stranded when the nucleic acid is at or below the melting temperature of the nucleic acid sequence.
11. The polymerase inhibitor of claim 1 wherein the nucleic acid portion of the polymerase inhibitor comprises DNA or a DNA mimetic.
12. The polymerase inhibitor of claim 1 wherein the nucleic acid comprises RNA or an RNA mimetic.
13. The polymerase inhibitor of claim 1 wherein the melting temperature of the double stranded portion of the nucleic acid portion of the polymerase inhibitor is in the range of about 25° C. to 80° C.
14. The polymerase inhibitor of claim 1 wherein the double stranded portion of the nucleic acid portion of the polymerase inhibitor is at least 10 bases in length.
15. A kit for inhibiting a nucleic acid polymerase comprising the polymerase inhibitor of claim 1 .
16. The kit of claim 15 further comprising one or more additional reagents for carrying out a nucleic acid amplification reaction.