Nucleic Acid Polymerase QC via Constant Specific Activity Dilution
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Solution Overview
Problem
Current methods for evaluating the activity and purity of nucleic acid polymerases in molecular diagnostic reagents are inadequate, particularly during intermediate stages of production, leading to difficulties in ensuring enzyme quality and consistency.
Innovation Solution
A method is developed to evaluate the state of nucleic acid polymerases by determining a dilution factor at which the specific activity exhibits a substantially constant value, allowing for accurate measurement of enzyme activity and purity through a nucleic acid polymerization reaction using a composition that includes a primer and a detectable signal.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If the product is measured without dilution, then the measurement reflects the actual concentration, but impurities and inactive proteins interfere with accurate activity measurement
Solution Approach 1:
The patent extracts the harmful components (impurities and inactive proteins) from the measurement system by diluting the product. This allows the active polymerase to be measured accurately while the interfering substances become negligible in concentration, resolving the contradiction between measuring actual concentration and avoiding interference.
Solution Approach 2:
The patent changes the concentration parameter by introducing a dilution factor. By measuring at multiple dilution levels and identifying where specific activity becomes constant, the method transforms the measurement from being concentration-dependent to being purity-independent, thereby achieving accurate specific activity measurement despite the presence of impurities.
2Measurement precision
If radioactive isotopes are used for measurement, then high accuracy is achieved, but environmental problems and health risks occur
Solution Approach 1:
The patent replaces expensive and harmful radioactive isotopes with a non-radioactive fluorescent detection system. The fluorescently labeled nucleotides provide sufficient measurement accuracy without the long-term environmental and health risks associated with radioactive materials, effectively substituting a harmful measurement approach with a safer alternative.
Solution Approach 2:
The patent substitutes the radioactive detection mechanism with a fluorescent detection mechanism. Instead of measuring radioactivity, the system uses fluorescence signals from labeled nucleotides incorporated during polymerization, replacing a harmful physical measurement approach with a safer optical detection method while maintaining measurement precision.
3Measurement precision
If dilution is applied to remove interference, then measurement accuracy improves, but the concentration of the enzyme decreases
Solution Approach 1:
The patent uses a feedback approach by measuring polymerase activity at multiple dilution levels and using the results to calculate the dilution factor where specific activity becomes constant. This feedback loop allows determination of accurate specific activity while accounting for the concentration reduction caused by dilution, transforming the concentration decrease into useful information for accurate measurement.
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
Enables precise monitoring of nucleic acid polymerase activity and purity throughout the preparation process, ensuring high-quality enzyme production by identifying a consistent specific activity level regardless of eluate type or impurity levels.
Implementation Method 1
DNA polymerase is an enzyme that polymerizes DNA using DNA or RNA as a template
Implementation Method 2
the primer is extended by the nucleic acid polymerase contained in the product under polymerization condition to form an extended duplex
Implementation Method 3
a detectable signal is provided in a manner dependent on the extended duplex
Data Source
Figure 1
Figure 2
Figure 3a~3b
AI summary
The present invention relates to a method for evaluating the state of a nucleic acid polymerase contained in a product obtained from the preparation process of a nucleic acid polymerase, and a method for obtaining a QC-able nucleic acid polymerase from the product. The present invention can evaluate the state of a nucleic acid polymerase contained in a product obtained from the preparation process of a nucleic acid polymerase by diluting the product obtained from the preparation process of the nucleic acid polymerase to a dilution factor at which specific activity of the nucleic acid polymerase contained in the product shows a substantially constant value.