DNA Concentration Measurement in Virus Capsids
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Solution Overview
Problem
Current methods for determining DNA concentration in DNA viruses like adeno-associated viruses (AAVs) and distinguishing between empty and full virus capsids are cumbersome, requiring labor-intensive purification processes and are not highly accurate due to interference from contaminants, which affects the precision and reproducibility of empty/full capsid ratios in gene therapy vector manufacturing.
Innovation Solution
The use of bio-layer interferometry (BLI) and fluorescent techniques to capture and quantify DNA from virus capsids, involving specific virus capture, lysis, and measurement of DNA concentration, allowing for the determination of empty versus full capsid ratios without the need for extensive sample purification, utilizing biosensor interferometer systems and probes with immobilized antibodies and proteins to measure wavelength shifts indicative of DNA content.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional purification methods are used to determine DNA concentration in AAV samples, then measurement accuracy is improved, but process complexity and time consumption increase significantly
Solution Approach 1:
The invention extracts and measures only the essential component (DNA concentration) directly from the crude AAV sample without requiring complete purification of all contaminants. By using real-time PCR to specifically detect and quantify DNA sequences, the method isolates the measurement of interest from the complex mixture, achieving accurate DNA concentration determination while bypassing the need for complex multi-step purification processes.
Solution Approach 2:
The invention introduces real-time PCR as an intermediary measurement technique that bridges the gap between crude sample analysis and accurate DNA quantification. The PCR process serves as a mediator that amplifies and specifically detects DNA sequences in the presence of other contaminants, enabling accurate measurement without requiring the sample to be fully purified first.
2Measurement precision
If traditional purification methods are used to determine DNA concentration in AAV samples, then measurement accuracy is improved, but time consumption increases
Solution Approach 1:
The invention performs preliminary DNA amplification and detection setup in advance using real-time PCR protocols, allowing for rapid quantification of DNA concentration directly from crude samples. By preparing the measurement system beforehand and using specific primers and probes, the method enables quick and accurate DNA concentration determination without time-consuming purification steps.
Solution Approach 2:
The invention replaces the mechanical and time-intensive purification processes with a biochemical detection system (real-time PCR). Instead of physically separating and purifying DNA through multiple centrifugation and filtration steps, the method uses molecular biology techniques to specifically detect and quantify DNA in the crude sample, dramatically reducing analysis time while maintaining accuracy.
3Measurement precision
If qPCR is used to determine AAV DNA content, then measurement precision is improved, but development and optimization time increases
Solution Approach 1:
The invention develops universal real-time PCR primers and probes that can detect and quantify DNA sequences across different AAV serotypes and vector configurations. This universal approach allows the same measurement protocol to be applied to various AAV-based gene therapy vectors without requiring separate optimization for each specific vector, thereby reducing development time while maintaining high measurement precision.
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 method provides a rapid, accurate, and reproducible means to determine DNA concentration and empty/full capsid ratios, reducing the complexity and time associated with traditional purification methods and enhancing the precision of gene therapy vector production.
Implementation Method 1
The use of bio-layer interferometry (BLI) and fluorescent techniques to capture and quantify DNA from virus capsids, involving specific virus capture, lysis, and measurement of DNA concentration, allowing for the determination of empty versus full capsid ratios without the need for extensive sample purification, utilizing biosensor interferometer systems and probes with immobilized antibodies and proteins to measure wavelength shifts indicative of DNA content.
Data Source
AI summary
The present invention is directed to a method of determining DNA concentration in a DNA virus. The invention features three basic steps. The initial step is the specific capture of a defined amount of virus capsid particles on a first solid phase. The second step is lysis of the capsid to release the virus DNA from the first solid phase into a lysis solution. After separating the lysis solution from the first solid phase, the third step is contacting the lysis solution with a second solid phase. The second solid phase captures total DNA derived from the captured capsid. The present invention is also directed to a method for measuring the percentage of full virus capsid, comprising first determining the ssDNA concentration in viruses, and then converting the ssDNA concentration to percentage of full virus capsid using a calibration curve having DNA concentration plotted against standards of % of full capsids.