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 method for inhibiting a nucleic acid polymerase, comprising performing a nucleic acid amplification reaction in the presence of a polymerase inhibitor, wherein the polymerase inhibitor comprises 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 method of claim 1 further comprising one or more additional polymerase inhibitors whose nucleic acid portions have different melting temperatures.
3. The method of claim 1 , wherein the polymerase inhibitor is present at a ratio of 1×10E-12 mol per unit of polymerase to 1×10E-10 mol per unit of polymerase.
4. The method of claim 1 , wherein the polymerase inhibitor is present in an amount that inhibits substantially all of the polymerase.
5. The method of claim 1 further comprising detecting the presence or absence of any nucleic acid produced by the nucleic acid amplification reaction.
6. The method of claim 5 further comprising quantifying the amount of any nucleic acid produced in the nucleic acid amplification reaction.
7. The method of claim 1 , wherein the polymerase inhibitor is not displaced from the polymerase enzyme by a primed target nucleic acid.
8. 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.
9. The method 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.
10. The method 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.
11. The method 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.
12. The method of claim 1 , wherein the nucleic acid portion of the polymerase inhibitor comprises DNA or a DNA mimetic.
13. The method of claim 1 , wherein the nucleic acid comprises RNA or an RNA mimetic.
14. The method 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.
15. The method of claim 1 , wherein the double stranded portion of the nucleic acid portion of the polymerase inhibitor is at least 10 bases in length.