Altered Polymerases for High-Throughput DNA Sequencing
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Solution Overview
Problem
Current polymerases with high DNA affinity limit their ability to incorporate nucleotides into multiple DNA templates in each reaction cycle, particularly in sequencing reactions where only a single nucleotide incorporation is required, leading to reduced reaction completion efficiency.
Innovation Solution
Development of altered polymerases with reduced DNA affinity, allowing them to form more productive polymerase-DNA complexes and incorporate nucleotides into multiple templates in each cycle, using nucleotide analogues with 3' sugar hydroxyl modifications that block further incorporation after each addition, enabling controlled nucleotide addition and efficient sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If polymerase has high DNA affinity, then it binds tightly to DNA template, but it limits ability to incorporate nucleotides into multiple DNA templates in each reaction cycle
Solution Approach 1:
The patent applies parameter changes by modifying the polymerase enzyme's DNA binding affinity through site-directed mutagenesis of residues in the C-terminal thumb subdomain. Specific amino acid substitutions (e.g., K844A, R878A, K800A in RB69 polymerase) reduce the strength of DNA binding interactions, allowing the polymerase to dissociate from and rebind to multiple DNA templates within a single reaction cycle, thereby improving reaction completion efficiency while maintaining sufficient binding stability for catalysis
2Stability of the object's composition
If polymerase binds tightly to DNA, then it maintains stable polymerase-DNA complex, but it reduces ability to form increased number of productive complexes in each reaction cycle
Solution Approach 1:
The patent implements dynamics by creating a polymerase with optimized binding kinetics - the mutated polymerase maintains sufficient DNA binding stability to form stable complexes for catalysis, but with reduced affinity that enables rapid dissociation and rebinding events. This dynamic behavior allows the same polymerase molecule to participate in multiple catalytic cycles with different DNA templates, increasing the number of productive complexes formed per reaction cycle while preserving complex stability during the catalytic step
Data Source
AI summary
Modified DNA polymerases have an affinity for DNA such that the polymerase has an ability to incorporate one or more nucleotides into a plurality of separate DNA templates in each reaction cycle. The polymerases are capable of forming an increased number of productive polymerase-DNA complexes in each reaction cycle. The modified polymerases may be used in a number of DNA sequencing applications, especially in the context of clustered arrays.