Mutant DNA polymerase(s) with improved strand displacement ability
Inventors
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-12286651-B2
Publication Date
2025-04-29
Expiration Date
Abstract
Disclosed are DNA polymerases having increased 5′-3′ strand displacement activity and substantially reduced 5′-3′ exonuclease and endonuclease activity relative to a corresponding, unmodified polymerase. The polymerases are useful in a variety of disclosed primer extension methods. Also disclosed are related compositions, including recombinant nucleic acids, vectors, and host cells, which are useful, e.g., for production of the DNA polymerases.
Core Innovation
The disclosed invention relates to a mutant DNA polymerase that shows increased 5′-3′ strand displacement activity and substantially reduced 5′-3′ exonuclease and endonuclease activity compared with a control DNA polymerase. The mutant DNA polymerase comprises an amino acid sequence at least 90% identical to SEQ ID NO:1 or SEQ ID NO:40 and includes a mutation corresponding to position 498 of SEQ ID NO:1.
The disclosed embodiments focus on thermostable mutants engineered to maintain strand displacement while suppressing 5′-3′ exonuclease and endonuclease activity, particularly at elevated temperatures used for PCR-like workflows. The content specifically describes multi-site mutant variants involving mutations at defined positions, including mutation combinations that include D498E together with other substitutions such as L524V, R598G, and M616I.
The content also describes related mutation sets involving other defined positions, including positions 580 and 709, and indicates that certain positions act as hot spots for improving the targeted activity profile. In addition to the mutant polymerase definition, the disclosed content describes related recombinant nucleic acids, vectors, and host cells comprising the engineered DNA polymerases.
The invention further documents use in primer extension and amplification workflows and describes kits and reaction mixtures that include the specified mutant polymerase and components suitable for amplification. The content also states that the workflows and reaction mixtures can be used in the presence of inhibitors such as hemoglobin/hemin and heparin, and provides assay results indicating enhanced strand displacement and reduced nuclease signals for selected mutants.
Claims Coverage
The disclosed claim set includes one independent claim directed to a mutant DNA polymerase having increased 5′-3′ strand displacement activity together with substantially reduced 5′-3′ exonuclease and endonuclease activity. The main inventive features are defined by sequence identity to SEQ ID NO:1/SEQ ID NO:40, a position-498-corresponding mutation, and a control polymerase with the SEQ ID NO:40 sequence; dependent claims further refine specific substitutions and broaden coverage to kits and reaction mixtures.
Increased strand displacement with reduced 5′-3′ exonuclease/endonuclease activity
A mutant DNA polymerase having increased 5′-3′ strand displacement activity and substantially reduced 5′-3′ exonuclease and endonuclease activity compared with a control DNA polymerase.
At least 90% identical sequence to SEQ ID NO:1 or SEQ ID NO:40
The amino acid sequence of the mutant DNA polymerase comprises an amino acid sequence at least 90% identical to SEQ ID NO:1 or SEQ ID NO:40.
Mutation corresponding to position 498 with control as SEQ ID NO:40
The mutant DNA polymerase comprises a mutation corresponding to position 498 of SEQ ID NO:1, and wherein the control DNA polymerase comprises the amino acid sequence of SEQ ID NO:40.
Overall, the claim coverage is anchored on a mutant DNA polymerase defined by increased 5′-3′ strand displacement activity together with substantially reduced 5′-3′ exonuclease and endonuclease activity, sequence similarity to SEQ ID NO:1/SEQ ID NO:40, and a position-498-corresponding mutation, with dependent refinements specifying particular substitutions and further coverage extending to kits and reaction mixtures.
Stated Advantages
Improved 5′-3′ strand displacement activity.
Substantially reduced 5′-3′ exonuclease and endonuclease activity.
Improved DNA-dependent activity and RT efficiency and mismatch tolerance, as stated in the disclosed content.
Reduced nuclease signals, as indicated by the disclosed assay results.
Usability with amplification workflows that can include elevated temperatures and inhibitors such as hemoglobin/hemin and heparin, as stated in the disclosed content.
Documented Applications
Primer extension/amplification workflows using the mutant DNA polymerase, as described in the disclosed content.
PCR-like workflows at elevated temperatures, as described in the disclosed content.
Isothermal amplification and related workflows such as SDA, LAMP, CSA, and PCDR, as indicated by the disclosed content.
Use in kits and reaction mixtures containing the mutant polymerase, with components for amplification such as primers, nucleoside triphosphates, and buffer, as described in the disclosed content.
Use of reaction mixtures in the presence of inhibitors such as hemoglobin/hemin and heparin, as described in the disclosed content.
Screening/assay workflows described in an example library generation and evaluation using oligos and a fluorescent reporter/quencher format, as described in the disclosed content.
Interested in licensing this patent?