Altered Polymerases for High-Fidelity Fast Sequencing
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Next-generation sequencing technologies face challenges in reducing sequencing time while maintaining high fidelity, as faster incorporation rates often compromise accuracy, leading to increased error rates and noisy signals due to phasing and pre-phasing issues.
Innovation Solution
Development of recombinant DNA polymerases with specific amino acid substitution mutations, such as those functionally equivalent to Tyr497, Phe152, Val278, Met329, Val471, Thr514, Leu631, and Glu734, which maintain high accuracy and reduce phasing and pre-phasing even at faster incorporation times, allowing for shorter sequencing run times without compromising fidelity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Speed
If the incorporation time is reduced to achieve faster sequencing, then the sequencing speed is improved, but the fidelity is worsened due to increased phasing rates, pre-phasing rates, and bypass rates
Solution Approach 1:
The patent applies parameter changes by modifying the chemical structure of nucleotide analogs through various substitutions (e.g., different sugar modifications, base modifications, and leaving group variations) to optimize the balance between incorporation speed and fidelity. Specific embodiments test multiple parameter variations including different reversible terminator groups, fluorophore attachments, and sugar ring modifications to achieve faster cycling without sacrificing accuracy
Solution Approach 2:
The patent implements dynamics by designing a system where the polymerase enzyme and nucleotide analogs work together in a dynamic equilibrium that allows rapid incorporation when conditions are optimal. The use of exchangeable reversible terminators and controlled nucleotide regeneration creates a dynamic system that can adapt to different sequencing cycle requirements while maintaining fidelity through the inherent proofreading capabilities of the polymerase
2Productivity
If the incorporation time is reduced to shorten sequencing run time, then the productivity is improved, but the error rate increases due to compromised fidelity
Solution Approach 1:
The patent employs parameter changes by systematically varying multiple chemical parameters of the nucleotide analogs including the type of reversible terminator, fluorophore selection, sugar modification patterns, and base analog variations. These parameter adjustments are optimized to enable faster nucleotide incorporation kinetics while maintaining sufficient chemical stability to prevent premature termination or incorrect base pairing, thereby reducing errors despite reduced incorporation time
Solution Approach 2:
The patent uses intermediary molecules such as exchangeable reversible terminators that mediate between the need for rapid incorporation and accurate base pairing. These intermediaries allow the polymerase to quickly add nucleotides while the chemical design of the terminators ensures they remain bound long enough to prevent further incorporation during the detection phase, thus maintaining fidelity despite faster cycling
3Loss of time
If the incorporation time is reduced to enable shorter sequencing cycles, then the cycle time is improved, but the signal quality deteriorates due to increased phasing and pre-phasing
Solution Approach 1:
The patent applies parameter changes by optimizing the chemical stability and binding affinity parameters of the nucleotide analogs. Specific modifications to the sugar ring, base, and terminator groups are designed to ensure that nucleotides incorporate rapidly but remain stably bound during the detection window. This optimization reduces phasing and pre-phasing effects by minimizing premature termination or extension events that would otherwise degrade signal quality in shorter cycles
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 altered polymerases achieve reduced error rates and improved sequencing quality metrics, including lower phasing and pre-phasing values, enabling faster sequencing cycles with longer read lengths and maintaining high fidelity, even at lower concentrations.
Implementation Method 1
recombinant DNA polymerases with specific amino acid substitution mutations... maintain high accuracy and reduce phasing and pre-phasing even at faster incorporation times
Data Source
AI summary
Presented herein are altered polymerase enzymes for improved incorporation of nucleotides and nucleotide analogues, in particular altered polymerases that maintain high fidelity under reduced incorporation times, as well as methods and kits using the same.