Altered Polymerases for Ambient Storage Stability
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Sequencing by synthesis methods face challenges with the stability of reagents at ambient temperatures, leading to degradation of modified nucleotides and increased pre-phasing, which reduces sequencing quality and read length.
Innovation Solution
Development of recombinant DNA polymerases with specific amino acid substitution mutations that improve resistance to incorporating degraded nucleotides without blocking groups, maintaining high incorporation rates of modified nucleotides while reducing incorporation of degraded ones.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If modified nucleotides are stored at ambient temperatures to reduce resource reliance, then ease of operation and cost are improved, but the stability of the nucleotides deteriorates leading to degradation and loss of blocking groups
Solution Approach 1:
The patent performs preliminary stabilization by engineering the polymerase enzyme with specific amino acid substitutions (e.g., at positions 408, 409, 410 in Tli Taq polymerase) that pre-adapt the enzyme to resist incorporating degraded nucleotides. This preliminary enzymatic adaptation allows the system to tolerate ambient storage conditions without requiring cold chain logistics.
Solution Approach 2:
The patent creates a modified copy of the natural polymerase enzyme through site-directed mutagenesis, generating engineered variants with altered specificity. These copied enzymes retain the ability to incorporate blocked nucleotides while gaining resistance to degraded forms, effectively decoupling storage requirements from enzyme performance.
2Productivity
If degraded nucleotides without blocking groups are incorporated, then productivity is temporarily improved through faster incorporation rates, but pre-phasing increases which reduces sequencing quality and read length
Solution Approach 1:
The patent applies local quality by creating heterogeneous polymerase populations with different specificities. The engineered polymerase maintains high incorporation rates for correctly blocked nucleotides (productive action) while simultaneously exhibiting reduced activity toward degraded nucleotides (quality control). This localized differentiation of enzymatic activity at the molecular level resolves the contradiction between speed and accuracy.
Solution Approach 2:
The patent changes the kinetic parameters of the polymerase enzyme through amino acid substitutions, altering its substrate specificity and incorporation kinetics. The engineered enzyme displays differential incorporation rates: fast for blocked nucleotides (maintaining productivity) and slow for degraded nucleotides (maintaining reliability), thereby resolving the trade-off between speed and quality.
3Device complexity
If standard polymerases are used with ambiently stored reagents, then device complexity is kept low, but pre-phasing increases due to recognition and incorporation of degraded nucleotides
Solution Approach 1:
The patent converts the harmful effect of ambient storage (nucleotide degradation) into a beneficial selection pressure for engineering more specific enzymes. By deliberately exposing the polymerase to degraded nucleotides during enzyme selection and optimization, the process generates polymerase variants that are inherently resistant to pre-phasing, thus converting the storage-induced harm into a quality improvement.
Data Source
AI summary
Presented herein are altered polymerase enzymes for improved incorporation of nucleotides and nucleotide analogues, in particular altered polymerases that maintain low pre-phasing rates when using ambiently stored polymerases, as well as methods and kits using the same.