Nucleic acid ligand inhibitors to DNA polymerases
AssigneeNeXstar Pharmaceuticals, Inc.
InventorsSumedha Jayasena, Larry Gold
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Abstract
This invention discloses high-affinity oligonucleotide ligands to the thermostable Taq polymerase, Tth polymerase and TZ05 polymerase. Specifically, this invention discloses DNA ligands having the ability to bind to the Taq, Tth and TZ05 polymerases and the methods for obtaining such ligands. The ligands are capable of inhibiting polymerases at any predetermined temperature.
Claims21
A method of identifying nucleic acid ligands to a polymerase, comprising:
- a) preparing a candidate mixture of nucleic acids;
- b) heating the candidate mixture of nucleic acids with said polymerase to a predetermined temperature, wherein nucleic acids having an increased affinity to the polymerase relative to the candidate mixture at that temperature may be partitioned from the remainder of the candidate mixture;
- c) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture at said predetermined temperature;
- and
- d) amplifying the increased affinity nucleic acids to yield a mixture of nucleic acids enriched for nucleic acid sequences with relatively higher affinity and specificity for binding to the polymerase, whereby nucleic acid ligands of the polymerase may be identified.
A method for inhibiting a DNA polymerase, comprising adding a nucleic acid ligand that inhibits said DNA polymerase DNA at a predetermined temperature to a DNA polymerization reaction.
A method for performing the Polymerase Chain Reaction (PCR) comprising:
- a) mixing a sample containing a nucleic acid sequence that is to be amplified with primers that are complementary to the sequences that flank the sequence to be amplified, a thermostable polymerase, and a nucleic acid ligand that is capable of inhibiting the polymerase at a predetermined temperature, yet allows the polymerase to be activated at temperatures above said predetermined temperature;
- and
- b) performing the standard PCR steps of melting the target nucleic acid, annealing the primers to the target nucleic acid, and synthesizing the target nucleic acid, by thermal cycling of the mixture.
A method of identifying a nucleic acid ligand switch for a polymerase, comprising:
- a) preparing a candidate mixture of nucleic acids;
- b) contacting the candidate mixture of nucleic acids with a polymerase, wherein nucleic acids having an increased affinity to the polymerase relative to the candidate mixture may be partitioned from the remainder of the candidate mixture;
- c) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture
- d) further partitioning the increased affinity nucleic acids of step c) based on their lack of affinity to the polymerase upon the variation of an environmental parameter;
- and
- e) amplifying the increased affinity nucleic acids obtained in d) to yield a mixture of nucleic acids enriched for nucleic acid sequences with relatively higher affinity and specificity for binding to the polymerase, whereby a nucleic acid ligand switch of the polymerase may be identified.
Dependencies are inferred from the claim text.