Modified Proteins for Ion-Based Sequencing Buffering Reduction
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Solution Overview
Problem
Ion-based nucleic acid sequencing methods face challenges in detecting hydrogen ions due to rapid diffusion and buffering effects of reaction components, which hinder efficient signal detection and increase sequencing errors.
Innovation Solution
Development of modified proteins with reduced buffering capacities, such as DNA polymerases with conservative amino acid substitutions, to minimize buffering effects and enhance ion detection in ion-based sequencing reactions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If proteins are used in ion-based sequencing reactions, then the reactions can proceed with proper enzymatic function, but the buffering effects of proteins hinder efficient detection of hydrogen ions
Solution Approach 1:
The patent applies parameter changes by modifying the buffering capacity of proteins through amino acid substitutions. Specifically, proteins are engineered to have reduced buffering capacity in the pH range of 6-8, which minimizes their interference with hydrogen ion detection while preserving their enzymatic function in sequencing reactions.
2Stability of the object's composition
If standard proteins with normal buffering capacity are used, then they maintain stable pH in reactions, but they reduce the signal from released hydrogen ions
Solution Approach 1:
The invention changes the buffering parameter of proteins by introducing amino acid substitutions that reduce buffering capacity in the critical pH range of 6-8. This allows the proteins to maintain sufficient pH stability for enzymatic function while minimizing their buffering effect on detected hydrogen ions, thereby improving signal detection.
3Stability of the object's composition
If proteins with high buffering capacity are used, then pH changes are minimized during reactions, but sequencing errors increase due to reduced ion detection accuracy
Solution Approach 1:
The patent modifies the buffering parameter of proteins to achieve an optimal balance. By reducing buffering capacity in the pH 6-8 range through amino acid substitutions, the proteins maintain adequate pH control for enzymatic activity while improving hydrogen ion detection accuracy, thereby reducing sequencing errors.
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 modified proteins improve signal detection and reduce sequencing errors by maintaining high accuracy and increasing the average length of error-free sequencing reads in ion-based nucleic acid sequencing.
Implementation Method 1
the buffering effects of proteins in an ion-based sequencing reaction can hinder the efficient detection of hydrogen ions
Implementation Method 2
The polymerase incorporates the appropriate nucleotide into the growing strand, and the hydrogen ion that is released changes the pH in the solution
Data Source
AI summary
Methods, compositions, systems, apparatuses and kits comprising modified proteins, particularly modified nucleic acid-binding proteins with altered buffering properties are provided. For example, in some embodiments, methods of forming modified proteins including one or more amino acid modifications to achieve desired pKa values are described. Furthermore, the invention provides methods for using such modified proteins in ion-producing reactions, such as ion-based nucleic acid sequencing reactions.