Nucleotide Purification via Phi29 Polymerase Incorporation
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Next-generation nucleic acid sequencing technologies face challenges with impurities in nucleotide solutions, leading to data corruption and increased costs due to purity requirements, which hinder the reuse of reagents and result in suboptimal sequencing quality.
Innovation Solution
A method involving the directed incorporation of nucleotides or nucleotide analogs into immobilized nucleic acid molecules, followed by their reuse to enhance solution purity and reduce phasing rates during sequencing, utilizing enzymes like phi29 polymerase for strand displacement and nucleotide incorporation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If high purity nucleotide solutions are used to maintain sequencing quality, then sequencing accuracy is improved, but reagent reuse is prevented and costs increase
Solution Approach 1:
The patent implements a two-stage nucleotide incorporation process where the first stage consumes nucleotides in a controlled manner to remove impurities, and the second stage recovers and reuses the purified nucleotide solution for actual sequencing. This allows the system to discard only the minimal amount needed for purification while recovering and reusing the bulk of the reagent, thereby maintaining sequencing accuracy while reducing reagent costs.
Solution Approach 2:
The patent performs preliminary nucleotide incorporation in a first reaction space before the actual sequencing reaction. This preliminary action consumes excess or impure nucleotides in advance, ensuring that only high-purity nucleotides remain for the subsequent sequencing step. By performing this purification action beforehand, the system ensures sequencing accuracy without needing to discard the entire reagent batch.
2Loss of substance
If nucleotide solutions are reused to reduce costs, then reagent cost decreases, but solution purity deteriorates and sequencing quality suffers
Solution Approach 1:
The patent divides the nucleotide incorporation process into two distinct segments: a first reaction space dedicated to purification through controlled incorporation, and a second reaction space for high-quality sequencing. This segmentation allows the system to handle impure and pure nucleotide populations separately, enabling reagent reuse while maintaining sequencing quality by ensuring that only purified nucleotides reach the sequencing stage.
Solution Approach 2:
The patent introduces an intermediary purification step between reagent reuse and sequencing. The first reaction space acts as an intermediary that processes the nucleotide solution, removing impurities through controlled incorporation reactions. This intermediary process enables the system to reuse nucleotide solutions while maintaining sequencing quality, as the intermediary step ensures purity requirements are met before sequencing occurs.
3Productivity
If impure nucleotide solutions are used to allow reagent reuse, then reagent cost decreases, but data corruption occurs and sequencing accuracy deteriorates
Solution Approach 1:
The patent changes the operational parameters of the nucleotide incorporation reaction in the first reaction space compared to the second reaction space. By adjusting parameters such as reaction conditions, enzyme concentrations, or incubation times in the first stage, the system optimizes for impurity removal rather than sequencing accuracy. This parameter change enables the system to process impure reagents safely in the first stage while protecting data quality in the second stage, thereby enabling reagent reuse without compromising reliability.
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
This approach improves reagent purity, reduces sequencing errors, and allows for the reuse of nucleotide solutions, thereby enhancing sequencing quality and reducing costs.
Implementation Method 1
utilizing enzymes like phi29 polymerase for strand displacement and nucleotide incorporation
Data Source
AI summary
The present disclosure provides methods and systems for processing a nucleotide mixture. A nucleotide mixture can be purified. A nucleotide mixture can be processed for use in nucleic acid synthesis. A nucleotide mixture can be processed for use in nucleic acid sequencing.