Engineered DNA Polymerase Variants for Thermostability and Fidelity
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Solution Overview
Problem
There is a need for thermostable DNA polymerases with high fidelity for applications such as PCR, as existing enzymes do not meet the required standards of stability and accuracy.
Innovation Solution
Engineered DNA polymerases with specific amino acid substitutions at defined positions in their polypeptide sequences, enhancing their thermostability and fidelity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If existing DNA polymerases are used, then PCR reactions can be performed, but the enzymes do not provide sufficient thermostability and fidelity
Solution Approach 1:
The patent applies parameter changes by introducing specific amino acid substitutions at defined positions in the DNA polymerase polypeptide sequence. These substitutions modify the enzyme's physical and chemical properties to achieve both high thermostability and high fidelity simultaneously, resolving the contradiction between these two parameters.
Solution Approach 2:
The engineered DNA polymerase combines amino acid residues with different properties at specific positions to create a composite structure that exhibits both thermostability and high fidelity. The substitution sets integrate multiple amino acid changes that work together to achieve the dual improvement in reliability and stability.
2Stability of the object's composition
If existing DNA polymerases are used, then PCR reactions can be performed, but the enzymes do not provide sufficient thermostability
Solution Approach 1:
The patent modifies the DNA polymerase structure through targeted amino acid substitutions that simultaneously improve both thermostability and fidelity. By changing specific parameters of the polypeptide sequence at defined positions, the enzyme achieves enhanced performance in both dimensions.
Solution Approach 2:
The engineered polymerase creates a composite amino acid structure where substituted residues work synergistically to provide both thermal stability and replication accuracy. The combination of multiple substitutions forms a composite functional unit that delivers dual improvements.
3Reliability
If amino acid substitutions are introduced to improve fidelity, then replication accuracy increases, but enzyme stability may be compromised
Solution Approach 1:
The patent applies local quality by introducing amino acid substitutions at specific, defined positions in the polypeptide sequence rather than throughout the entire structure. This localized modification approach allows fidelity improvement through targeted changes while preserving overall structural stability through careful selection of substitution positions and types.
Solution Approach 2:
The engineered polymerase uses controlled parameter changes at specific positions to improve fidelity without compromising stability. The substitution sets are designed to modify local properties that affect fidelity while maintaining global structural integrity.
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 engineered DNA polymerases exhibit improved thermostability and fidelity, making them suitable for reliable use in PCR and other diagnostic applications.
Implementation Method 1
DNA polymerases are enzymes that synthesize DNA from deoxyribonucleotides
Data Source
AI summary
The present invention provides engineered DNA polymerase polypeptides and compositions thereof, as well as polynucleotides encoding the engineered DNA polymerase polypeptides. The invention also provides methods for use of the compositions comprising the engineered DNA polymerase polypeptides for diagnostic and other purposes.