Pyrococcus Polymerase Mutations for Modified Nucleotide Fidelity
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Solution Overview
Problem
Current DNA polymerases struggle with insufficient incorporation rates and fidelity of modified nucleotides, leading to errors in sequencing applications due to residual modifications and inappropriate nucleotide incorporation.
Innovation Solution
Modified Archaeal family B polymerases derived from Pyrococcus, with specific amino acid sequences, are developed to enhance the incorporation of modified nucleotides, improving sequencing accuracy and efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If current DNA polymerases are used, then the sequencing process can proceed, but the incorporation rate of modified nucleotides is insufficient and misincorporation errors occur
Solution Approach 1:
The patent applies parameter changes by modifying the amino acid sequence of the DNA polymerase enzyme. Specifically, it introduces mutations at positions 408, 409, and 410 (e.g., changing residues to alanine, glycine, or serine) to alter the enzyme's catalytic properties. These parameter changes in the enzyme's structure enable improved incorporation kinetics of modified nucleotides while maintaining or enhancing sequencing fidelity, thereby resolving the contradiction between productivity and reliability.
2Productivity
If DNA polymerase is modified to increase incorporation rates of nucleotide analogues, then productivity improves, but fidelity may decrease due to promiscuous incorporation
Solution Approach 1:
The patent applies local quality by making targeted, localized modifications to specific amino acid residues (positions 408, 409, 410) in the polymerase active site rather than global changes. This localized approach allows the enzyme to selectively improve incorporation of desired modified nucleotides while maintaining discrimination against incorrect nucleotides, thus achieving both high productivity and high manufacturing precision simultaneously.
3Productivity
If modifications are made to improve modified nucleotide incorporation, then productivity increases, but the enzyme may become too promiscuous and incorporate inappropriate nucleotides
Solution Approach 1:
The patent applies parameter changes by systematically modifying amino acid residues in the polymerase active site (positions 408, 409, 410) to optimize the enzyme's catalytic parameters. The specific mutations (e.g., to alanine, glycine, or serine) tune the enzyme's kinetics to achieve rapid incorporation of modified nucleotides with half times suitable for high-throughput sequencing while maintaining fidelity through preserved nucleotide selectivity mechanisms.
Data Source
AI summary
Provided herein are modified Archaeal family B polymerases derived from species of the Archaeal microorganism Pyrococcus that exhibit improved incorporation of nucleotide analogues utilized in DNA sequencing.