Mutated Sequencing Polymerases for Fidelity and Terminator Incorporation
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Solution Overview
Problem
Native DNA polymerases exhibit discriminatory behavior against modified nucleotides, necessitating a balance between incorporating reversible terminators and maintaining high fidelity, which is challenging for sequencing applications.
Innovation Solution
A polymerase with an amino acid sequence at least 80% identical to SEQ ID NO: 1, featuring mutations at specific positions like 306, to accommodate modified nucleotides while preserving fidelity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If native DNA polymerases are used for sequencing applications with reversible terminators, then high fidelity is maintained, but incorporation efficiency of modified nucleotides is poor
Solution Approach 1:
The patent applies parameter changes by introducing specific amino acid mutations at position 306 (and other positions) in the polymerase sequence. These mutations alter the enzyme's biochemical parameters to accommodate modified nucleotides with reversible terminators, enabling efficient incorporation while maintaining fidelity through controlled changes rather than complete redesign
2Productivity
If mutations are introduced in the polymerase to accommodate reversible terminators, then incorporation kinetics are improved, but misincorporation error increases
Solution Approach 1:
The patent applies local quality by making targeted mutations only at specific positions (notably position 306) in the polymerase sequence rather than broadly altering the entire enzyme. This localized approach modifies the active site to accommodate reversible terminators while preserving the overall fidelity characteristics of the polymerase through conservative changes
3Adaptability or versatility
If the polymerase is altered to accept modified substrates, then adaptability is improved, but fidelity is compromised
Solution Approach 1:
The patent applies segmentation by separating the functions of substrate acceptance and fidelity maintenance into distinct functional regions of the polymerase. The mutations at position 306 modify the active site for substrate acceptance, while other regions maintain fidelity through conserved structural and functional elements, allowing the enzyme to handle both native and modified substrates with appropriate specificity
Data Source
AI summary
Disclosed herein, inter alia, are polymerases designed for accurate incorporation of nucleotides into a primer bound to a template polynucleotide.