Mutated Polymerase for Isothermal Strand Displacement
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Solution Overview
Problem
Current nucleic acid amplification techniques, such as PCR, require thermal cycling and rely on thermostable strand-displacing enzymes like SD DNA polymerase and Bst DNA polymerase, which are inactivated at elevated temperatures, limiting efficient isothermal amplification.
Innovation Solution
Development of a polymerase with specific amino acid mutations, including at positions 588, 97, and 742, that enhances strand displacement activity without thermal cycling, enabling continuous replication and amplification at elevated temperatures.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If SD DNA polymerase or Bst DNA polymerase is used for isothermal amplification, then strand displacement activity is achieved, but the enzyme is inactivated at elevated temperatures
Solution Approach 1:
The patent applies parameter changes by introducing specific amino acid mutations at positions 588, 97, and 742 in the polymerase sequence. These mutations alter the enzyme's physical and chemical properties to enhance thermostability while preserving strand displacement activity, enabling the enzyme to function reliably at elevated temperatures for isothermal amplification
2Productivity
If thermal cycling is used for DNA amplification, then efficient amplification is achieved, but the process becomes complex and time-consuming
Solution Approach 1:
The patent enables continuous useful action by creating a polymerase that performs both strand displacement and maintains stability at elevated temperatures in a single continuous isothermal reaction. This eliminates the need for repeated heating and cooling cycles, allowing amplification to proceed continuously at one temperature, thereby simplifying the process and reducing time
Solution Approach 2:
Instead of using thermal cycling to achieve strand separation and primer annealing, the patent inverts the approach by using a mutant polymerase with enhanced strand displacement activity that can perform these functions isothermally, replacing the thermal cycling mechanism with an enzymatic mechanism
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The mutated polymerase facilitates efficient nucleic acid amplification by maintaining activity at elevated temperatures, overcoming the limitations of existing thermostable enzymes and improving isothermal amplification processes.
Implementation Method 1
contacting the primer-template hybridization complex with a DNA polymerase and nucleotides, wherein the DNA polymerase is the polymerase as described herein; and subjecting the primer-template hybridization complex to conditions which enable the polymerase to incorporate one or more nucleotides into the primer-template hybridization complex
Data Source
AI summary
Disclosed herein, inter alia, are novel strand-displacing polymerases and methods of use thereof.