Thermococcus Polypeptide for High-Fidelity Long DNA Amplification
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Solution Overview
Problem
Current DNA polymerases used in genetic engineering and PCR methods face challenges such as low extension velocity, reduced fidelity, and difficulty in amplifying long DNA chains due to strong exonuclease activity, limiting their suitability for high-fidelity applications.
Innovation Solution
A novel polypeptide with high-fidelity DNA polymerase activity is derived from a hyperthermophilic archaebacterium of the genus Thermococcus, specifically characterized by amino acid sequences that enhance primer extension and processivity, allowing for accurate amplification of long DNA chains with reduced error rates.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If Pfu-derived DNA polymerase is used for PCR, then high fidelity is achieved due to 3'→5' exonuclease activity, but amplification time increases and long chain amplification becomes difficult due to low extension velocity and processivity
Solution Approach 1:
The patent introduces specific amino acid substitutions (Phe629→Leu, Ala630→Ser, Asp632→Gly, Asp632→Asn, Asp632→Cys, Asp632→Val, Thr634→Ala, Gln637→Pro, Asp640→Gly, Asn641→Ser, Asn641→Thr, Asn641→Ala, Asn641→Val, Asn641→Cys, Asn641→Phe, Asn641→Leu, Asn641→Ile, Asn641→Met, Asn641→Gln, Asn641→Arg, Asn641→His, Asn641→Lys, Asn641→Ser, Asn641→Thr, Asn641→Ala, Asn641→Val, Asn641→Cys, Asn641→Phe, Asn641→Leu, Asn641→Ile, Asn641→Met, Asn641→Gln, Asn641→Arg, Asn641→His, Asn641→Lys) at the exonuclease active site to reduce exonuclease activity while preserving polymerase activity, thereby improving extension velocity and processivity while maintaining high fidelity
2Productivity
If KOD DNA polymerase is used for PCR, then high extension velocity and processivity are achieved, but fidelity decreases and primer/amplification product degradation occurs due to strong 3'→5' exonuclease activity
Solution Approach 1:
The patent applies the same amino acid substitution strategy at the exonuclease active site to reduce the overly strong 3'→5' exonuclease activity of KOD DNA polymerase, thereby preventing primer and amplification product degradation while maintaining high extension velocity and processivity
3Reliability
If KOD-Plus-DNA polymerase is used for hot-start PCR, then polymerase activity and 3'→5' exonuclease activity are suppressed at normal temperature, but extension velocity decreases to levels equivalent to Pfu-derived DNA polymerase
Solution Approach 1:
The patent uses amino acid substitutions to modify the enzyme's intrinsic properties, eliminating the need for antibody-based suppression while achieving both hot-start capability and high extension velocity through direct enzymatic property modification
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 novel polypeptide enables high-fidelity DNA amplification with improved extension velocity and processivity, suitable for cloning and sequencing, even in multiple PCR cycles, and is effective in amplifying DNA fragments up to 15 kilobase pairs without the need for additional enzymes.
Implementation Method 1
DNA polymerases are enzymes that are useful as reagents for genetic engineering and widely utilized for DNA sequencing, labeling, site-directed mutagenesis and the like
Implementation Method 2
It has a 3′→5′ exonuclease activity, which is known as a proofreading activity, and it exhibits relatively high fidelity among thermostable enzymes
Data Source
AI summary
A polypeptide having a high fidelity DNA polymerase activity and thus being useful as a genetic engineering reagent; a gene encoding this polypeptide; a method of producing the polypeptide; and a method of amplifying a nucleic acid by using the polypeptide.