DNA Polymerase Mutants for Modified Nucleotide Sequencing
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Solution Overview
Problem
Current sequencing polymerases, particularly thermostable family B polymerases from hyperthermophilic archaea, exhibit low polymerization capability for incorporating artificially modified nucleotides or nucleotide analogs, leading to sequencing speed and quality issues in sequencing-by-synthesis methods.
Innovation Solution
Engineered DNA polymerase mutants with specific amino acid mutations at positions 409, 410, and 486 of Pyrococcus abyssi DNA polymerase enhance the incorporation efficiency of non-natural dNTPs, improving sequencing speed and quality.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If thermostable family B polymerases from hyperthermophilic archaea are used for sequencing-by-synthesis, then thermostability and DNA polymerase activity are maintained, but the polymerization capability for incorporating artificially modified nucleotides or nucleotide analogs is low
Solution Approach 1:
The patent applies parameter changes by mutating specific amino acid residues at positions 409, 410, and 486 in the polymerase active site. These parameter changes in the enzyme's structure enable it to accommodate and incorporate modified nucleotides with fluorescent labels and reversible terminators, thereby improving polymerization capability while preserving thermostability and catalytic activity
Solution Approach 2:
The patent applies local quality by making targeted amino acid substitutions specifically at positions 409, 410, and 486 in the active site region, while leaving the rest of the polymerase structure unchanged. This localized modification approach allows the enzyme to gain enhanced ability to incorporate modified nucleotides while maintaining its overall thermostable structure and DNA polymerase function
2Productivity
If amino acid mutations are introduced at positions 409, 410, and 486 to enhance incorporation efficiency, then sequencing speed and quality improve, but enzyme structure complexity increases
Solution Approach 1:
The patent introduces only three specific amino acid mutations at positions 409, 410, and 486 in the active site, leaving the rest of the enzyme structure unchanged. This minimal local modification approach achieves improved sequencing performance without significantly increasing overall enzyme structure complexity
Solution Approach 2:
The patent changes specific amino acid parameters at three key positions to optimize the active site for modified nucleotide incorporation. These targeted parameter changes achieve enhanced sequencing speed and quality while maintaining a relatively simple enzyme structure with only three substitutions
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 mutants significantly improve the incorporation efficiency of non-natural dNTPs, enhancing sequencing speed and quality in sequencing-by-synthesis methods.
Implementation Method 1
DNA polymerase mutant, a nucleic acid molecule, an expression vector, a recombinant cell, a recombinant strain, a method for producing DNA polymerase mutant, a complex, a method for nucleic acid sequencing
Data Source
Figure 1
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.