Recombinant KOD Polymerase Mutations for Sequencing Read Length
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Solution Overview
Problem
Current SBS sequencing technologies face limitations such as short read length and slow reaction rates due to the 3′-5′ exonuclease activity of wild-type KOD polymerase, which hinders efficient DNA replication and sequencing processes.
Innovation Solution
Modification of specific amino acid residues in the KOD DNA polymerase at positions like 675, 385, 710, and others, along with the addition of a His tag, to create recombinant KOD polymerase variants that enhance DNA polymerase activity, improving catalytic efficiency and reaction rates for DNA sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If wild-type KOD polymerase is used for SBS sequencing, then DNA replication can be performed, but the read length is short and reaction rate is slow due to 3'-5' exonuclease activity
Solution Approach 1:
The patent removes the 3'-5' exonuclease activity from KOD polymerase through site-directed mutagenesis, specifically mutating residues in the exonuclease domain (e.g., E385Q, E675Q mutations). This extraction of the harmful exonuclease function while preserving the polymerase activity resolves the contradiction by eliminating the source of sequencing errors and read length limitations without sacrificing DNA replication capability
Solution Approach 2:
The patent modifies specific amino acid parameters in the polymerase structure, particularly in the exonuclease domain and finger domain. By changing residues like E385 to Q, E675 to Q, and optimizing dNTP binding site residues, the enzyme's kinetic parameters are improved, achieving faster reaction rates and longer read lengths while maintaining fidelity
2Productivity
If multiple amino acid residues are modified to improve polymerase activity, then reaction rate increases, but enzyme structure complexity increases
Solution Approach 1:
The patent applies local quality changes by specifically mutating only the critical residues in the exonuclease domain and dNTP binding site, rather than modifying the entire enzyme structure. Key mutations include E385Q, E675Q in the exonuclease domain and specific residues in the finger domain. This localized approach improves polymerase activity while minimizing structural complexity increases
Solution Approach 2:
The patent creates a multi-functional polymerase variant that simultaneously achieves: (1) elimination of 3'-5' exonuclease activity, (2) enhanced polymerase activity, (3) improved processivity for longer read lengths, and (4) maintained thermostability. This universal optimization through coordinated mutations resolves the contradiction by making the enzyme structure serve multiple improved functions
Data Source
AI summary
Provided is a recombinant KOD polymerase, which is the following A) or B): the polymerase shown in A) is a protein having DNA polymerase activity that is obtained by modifying amino acid residues in at least one of the following 18 positions in a wild-type KOD DNA polymerase amino acid sequence: 675th, 385th, 710th, 674th, 735th, 736th, 606th, 709th, 347th, 349th, 590th, 676th, 389th, 589th, 680th, 384th, 496th and 383rd; the polymerase described by B) is a protein having DNA polymerase activity that is derived from A) by adding a tag sequence to an end of the amino acid sequence of the protein shown in A).