Recombinant KOD Polymerase Mutants for Sequencing Read Length
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Solution Overview
Problem
Current SBS sequencing technologies face limitations in reaction rate and read length, resulting in slow processing times and short sequencing outputs.
Innovation Solution
Development of recombinant KOD DNA polymerase mutants with specific amino acid modifications in key positions, enhancing catalytic and physical properties for improved nucleotide incorporation efficiency, specifically targeting positions 267, 326, 347, 353, 375, 378, 379, 380, 451, 452, 453, 454, 457, 461, 465, 470, 474, 477, 478, 479, 480, 482, 484, 485, 486, 493, 496, 497, 514, 574, 584, 605, 610, 630, 665, 666, 667, 674, 676, 680, 682, 698, 707, 718, 723, and 729, to accelerate biochemical reactions and extend sequencing capabilities.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If nucleotides with 3′ blocking groups are used in SBS sequencing, then controlled nucleotide incorporation is achieved, but reaction rate decreases and read length shortens
Solution Approach 1:
The patent applies parameter changes by modifying amino acid residues at specific positions (267, 326, 347, 349, 353, 375, 378, 379, 380, 385, 451, 452, 453, 454, 457, 461, 465, 470, 474, 477, 478, 479, 480, 482, 484, 485, 486, 493, 496, 497, 514, 574, 584, 605, 610, 630, 665, 666, 667, 674, 676, 680, 682, 698, 707, 718, 723, 729) in the KOD polymerase structure. These amino acid substitutions alter the enzyme's catalytic properties and steric environment, enabling it to accommodate 3′ blocking group nucleotides while maintaining high reaction rates and extending read lengths beyond the limitations of wild-type polymerases.
2Reliability
If nucleotides with 3′ blocking groups are used in SBS sequencing, then controlled nucleotide incorporation is achieved, but read length shortens
Solution Approach 1:
The patent applies parameter changes by modifying amino acid residues at specific positions (267, 326, 347, 349, 353, 375, 378, 379, 380, 385, 451, 452, 453, 454, 457, 461, 465, 470, 474, 477, 478, 479, 480, 482, 484, 485, 486, 493, 496, 497, 514, 574, 584, 605, 610, 630, 665, 666, 667, 674, 676, 680, 682, 698, 707, 718, 723, 729) in the KOD polymerase structure. These amino acid substitutions alter the enzyme's catalytic properties and steric environment, enabling it to accommodate 3′ blocking group nucleotides while maintaining high reaction rates and extending read lengths beyond the limitations of wild-type polymerases.
3Reliability
If KOD DNA polymerase is used for SBS sequencing, then heat resistance and accuracy are improved, but reaction rate with modified nucleotides is limited
Solution Approach 1:
The patent applies parameter changes by systematically modifying amino acid residues at 48 specific positions in the KOD polymerase structure. These substitutions optimize the enzyme's active site geometry and catalytic mechanism to better accommodate modified nucleotides with 3′ blocking groups, thereby increasing reaction rates while preserving the heat resistance and sequencing accuracy that characterize the KOD polymerase family.
Solution Approach 2:
The patent applies local quality by making targeted amino acid substitutions at specific positions (267, 326, 347, 349, 353, 375, 378, 379, 380, 385, 451, 452, 453, 454, 457, 461, 465, 470, 474, 477, 478, 479, 480, 482, 484, 485, 486, 493, 496, 497, 514, 574, 584, 605, 610, 630, 665, 666, 667, 674, 676, 680, 682, 698, 707, 718, 723, 729) rather than globally altering the polymerase structure. This localized modification approach preserves the overall heat-resistant properties and fidelity of the KOD polymerase while optimizing specific regions involved in nucleotide binding and catalysis for improved reaction rates with modified substrates.
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 recombinant KOD DNA polymerase mutants demonstrate increased reaction rates and catalytic efficiency, leading to longer read lengths and faster sequencing times compared to the original KOD DNA polymerase GH78, thereby enhancing the overall sequencing process.
Implementation Method 1
DNA polymerase has a function of replicating DNA quickly and accurately
Implementation Method 2
The use of nucleotides with 3′ blocking groups allows the incorporation of nucleotides into the polynucleotide chain in a controlled manner
Data Source
AI summary
Provided is a recombinant KOD polymerase. A KOD polymerase mutant is a protein as follows: the protein is a protein having DNA polymerase activity that is obtained by modifying amino acid residues in at least one of the following 48 positions in the amino acid sequence of KOD DNA polymerase GH78: 267, 326, 347, 349, 353, 375, 378, 379, 380, 385, 451, 452, 453, 454, 457, 461, 465, 470, 474, 477, 478, 479, 480, 482, 484, 485, 486, 493, 496, 497, 514, 574, 584, 605, 610, 630, 665, 666, 667, 674, 676, 680, 682, 698, 707, 718, 723 and 729, without changing other amino acid sequences; and compared with KOD DNA polymerase GH78, with regard to catalysis, the recombinant DNA polymerase exhibits a faster reaction rate, better catalytic efficiency, better affinity and other advantages, thereby improving the reaction rate of DNA polymerase in sequencing and increasing the reaction read length.