Chimeric DNA Polymerase Resolving Fidelity-Processivity Trade-off
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Solution Overview
Problem
Current DNA polymerases, despite their correcting functions, suffer from relatively low processivity, which results in reduced DNA amplification yields, and those with high processivity often compromise on fidelity, making them unsuitable for applications requiring high accuracy.
Innovation Solution
A chimeric DNA polymerase is created by combining heterologous domains from different DNA polymerases, such as Pyrococcus furiosus, Thermococcus kodacaraensis, and Pyrococcus abyssi, to achieve high processivity, elongation rate, thermal stability, and salt resistance, while maintaining high fidelity, thereby enhancing DNA amplification, synthesis, and sequencing capabilities.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If DNA polymerases with correcting function are used, then fidelity is improved, but processivity deteriorates
Solution Approach 1:
The patent merges functional domains from different DNA polymerases to create a chimeric enzyme. Specifically, it combines the high-fidelity exonucleolytic domain from Family B polymerases (e.g., Pfu, Pab) with the high-processivity domains from Family A polymerases (e.g., Taq, Tth), resulting in a chimeric DNA polymerase that simultaneously achieves both high fidelity and high processivity, resolving the contradiction between these two properties
Solution Approach 2:
The chimeric DNA polymerase is constructed as a composite protein entity with domains from different sources. The enzyme comprises a Family A polymerase backbone (providing processivity) fused with Family B exonucleolytic domains (providing fidelity), creating a composite biological material that exhibits properties superior to individual parent enzymes
2Productivity
If Family A DNA polymerases are used, then processivity is improved, but fidelity deteriorates
Solution Approach 1:
The patent merges the high-processivity domains from Family A polymerases with the high-fidelity exonucleolytic domains from Family B polymerases. This combination allows the chimeric enzyme to inherit the processivity advantage of Family A enzymes while gaining the fidelity advantage of Family B enzymes, thus resolving the contradiction
3Reliability
If Family B DNA polymerases are used, then fidelity is improved, but processivity deteriorates
Solution Approach 1:
The patent combines the high-fidelity exonucleolytic domains from Family B polymerases with the high-processivity domains from Family A polymerases. This cross-family merging strategy enables the chimeric enzyme to achieve both high fidelity (from Family B) and high processivity (from Family A), resolving the limitation of Family B enzymes alone
Data Source
AI summary
A chimeric DNA polymerase includes: a first fragment having at least 80% homology to at least part of an N-end domain of KOD DNA polymerase; a second fragment having at least 80% homology to at least part of an exonucleolytic domain of Pab DNA polymerase; a third fragment having at least 80% homology to at least part of the N-end domain of KOD DNA polymerase; a fourth fragment having at least 80% homology to at least part of a palm domain of Pfu DNA polymerase; a fifth fragment having at least 80% homology to at least part of a finger domain of Pab DNA polymerase; a sixth fragment having at least 80% homology to at least part of the palm domain of Pfu DNA polymerase; and a seventh fragment having at least 80% homology to at least part of a thumb domain of KOD DNA polymerase.