Protease Treatment for Polymerase Enzyme Complex Enrichment
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Solution Overview
Problem
Current methods for single molecule nucleic acid sequencing face challenges in throughput and yield due to the presence of free polymerases, which increase background noise and reduce accuracy by binding to fluorescent substrates and competing for space in reaction regions.
Innovation Solution
A method involving a loading composition enriched for polymerase enzyme complexes using a protease to remove free polymerases, which includes applying a protease to a composition containing polymerase enzyme complexes, non-catalytic metal ions, and nucleotides, followed by distribution onto a substrate, to enhance sequencing data accuracy and minimize background noise.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If free polymerases are present in the loading composition, then the polymerase enzyme can bind to templates and perform sequencing reactions, but background noise increases and sequencing accuracy decreases
Solution Approach 1:
The patent applies protease to selectively remove free polymerases from the loading composition through proteolytic cleavage. This extraction of the harmful component (free polymerase) eliminates its ability to bind fluorescent substrates and generate background noise, thereby improving sequencing accuracy while preserving polymerase enzyme complexes that are protected from protease digestion
Solution Approach 2:
The patent introduces protease as an intermediary substance that mediates the selective removal of free polymerases. The protease acts as a tool to differentiate between free polymerases (to be removed) and polymerase enzyme complexes (to be preserved), enabling selective elimination of the harmful factor without affecting the functional complexes
2Productivity
If free polymerases are present in the loading composition, then sufficient polymerase activity is available for sequencing reactions, but the polymerases compete for space in reaction regions and reduce throughput
Solution Approach 1:
By removing free polymerases through protease treatment, the patent eliminates the competition for space in reaction regions. This extraction increases the effective concentration of functional polymerase enzyme complexes in the loading composition, thereby improving throughput and yield of sequencing reactions
3Reliability
If protease is applied to remove free polymerases, then sequencing accuracy improves, but the loading composition requires purification to remove protease and its byproducts
Solution Approach 1:
The patent performs protease treatment on the loading composition before distribution to the substrate. This preliminary action allows for centralized purification steps to be performed on the bulk composition rather than on individual reaction regions, reducing overall process complexity. The purification can be performed once for the entire loading composition, eliminating the need for repeated purification at each sequencing location
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 enrichment of polymerase enzyme complexes improves sequencing accuracy and signal-to-noise ratio by reducing background noise from free polymerases, leading to more reliable and efficient nucleotide sequence determination.
Implementation Method 1
applying a protease to the loading composition to remove free polymerases
Data Source
AI summary
The present invention provides methods, compositions, and systems for enriching compositions for polymerase enzyme complexes. In particular, the methods, compositions, and systems of the present invention remove free polymerases from the compositions using one or more purification steps, including protease treatment, thus enriching the compositions for polymerases complexed with a template nucleic acid.