Sequencing Polymerase Mutants for Modified dNTP Incorporation
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Solution Overview
Problem
Current sequencing polymerases have low polymerization capability to incorporate artificially modified nucleotides or nucleotide analogs, leading to sequencing speed and quality issues in sequencing-by-synthesis methods.
Innovation Solution
Enzyme engineering modifications are applied to the active sites of thermostable family B polymerases from hyperthermophilic archaea, creating DNA polymerase mutants with specific amino acid mutations at positions 409, 410, and 486, enhancing the incorporation efficiency of non-natural dNTPs.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If natural polymerases are used for sequencing-by-synthesis, then the sequencing process can be performed, but the polymerization capability to incorporate artificially modified nucleotides or nucleotide analogs is low
Solution Approach 1:
The patent applies parameter changes by mutating specific amino acid residues in the polymerase enzyme to alter its catalytic properties. The mutations at positions Q409, N411, and Y486 change the enzyme's parameters to enhance both its ability to incorporate modified nucleotides and its polymerization speed, resolving the contradiction between adaptability and productivity.
Solution Approach 2:
The patent applies local quality by making targeted mutations at specific locations (active site residues) within the polymerase enzyme rather than modifying the entire enzyme globally. This localized modification approach allows the enzyme to gain enhanced capability for incorporating modified nucleotides while maintaining overall enzyme function and high polymerization capability.
2Speed
If existing natural polymerases are used, then the sequencing process can proceed, but the sequencing speed is limited due to low polymerization capability
Solution Approach 1:
The patent uses parameter changes by mutating residues Q409, N411, and Y486 to alter the enzyme's kinetic parameters. These mutations enhance the polymerization rate, which directly increases sequencing speed while maintaining high productivity, thus resolving the contradiction between speed and productivity.
3Reliability
If existing natural polymerases are used, then the sequencing process can be performed, but the sequencing quality is affected due to low polymerization capability
Solution Approach 1:
The patent applies parameter changes through targeted mutations that improve the enzyme's fidelity and incorporation efficiency of modified nucleotides. By changing the parameters of residues at positions Q409, N411, and Y486, the enzyme achieves both high sequencing quality and maintained productivity.
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 DNA polymerase mutants improve sequencing speed and quality by effectively incorporating non-natural dNTPs, particularly those with fluorescent labels, in sequencing-by-synthesis methods.
Implementation Method 1
capable of incorporating specific non-natural dNTPs, thereby improving sequencing speed and quality in sequencing-by-synthesis (SBS) methods
Data Source
AI summary
Provided is a DNA polymerase mutant for sequencing. The DNA polymerase mutant includes: compared with a Pyrococcus abyssi DNA polymerase exo-mutant, at least three amino acid mutations in the following four sites or functionally equivalent sites: position 409, position 410, position 411, and position 486. The Pyrococcus abyssi DNA polymerase exo-mutant has an amino acid sequence as set forth in SEQ ID NO: 1.