Nucleic Acid Variation Quantification via Merged PCR Assay
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Solution Overview
Problem
Conventional methods for determining DNA methylation levels are labor-intensive, time-inefficient, and prone to errors due to the need for constructing multiple standard curves and handling large DNA samples, with well-to-well variations introducing significant measurement errors.
Innovation Solution
A method that calculates the percentage of variant nucleic acid by determining the ratio of variant to non-variant copies using quantitative amplification data from a sample, eliminating the need for external standards and reducing the complexity of DNA handling through PCR+INVADER assays, which allow simultaneous amplification and detection of methylated and unmethylated DNA in the same reaction vessel.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional methods use multiple external standard curves to determine DNA methylation levels, then measurement accuracy can be maintained, but the process becomes labor-intensive and time-inefficient
Solution Approach 1:
The patent combines the quantification of methylated and unmethylated DNA into a single real-time PCR reaction by using methylation-specific primers that can amplify both forms simultaneously, with distinct fluorescent labels allowing differentiation. This eliminates the need for separate standard curves for each DNA form, reducing the number of reactions from multiple PCRs to a single reaction while maintaining measurement accuracy through ratio-based calculation.
Solution Approach 2:
The invention creates a universal quantification method that uses a single standard curve applicable to both methylated and unmethylated DNA detection. The methylation-specific primers and fluorescent labeling system enable one reaction mixture to serve multiple detection purposes, eliminating the need for separate external standards for each DNA form and significantly reducing the time and labor required.
2Measurement precision
If conventional methods construct multiple standard curves from several external standard PCRs, then quantitative accuracy is achieved, but the device complexity and number of reactions increase
Solution Approach 1:
The patent merges the detection of methylated and unmethylated DNA into a single real-time PCR reaction system. By using methylation-specific primers that bind to both methylated and unmethylated forms at different positions, and incorporating fluorescent labels on the primers themselves, the system generates distinct fluorescent signals from one reaction, eliminating the need for multiple separate reactions and standard curves.
Solution Approach 2:
The invention enables the reaction mixture to self-differentiate between methylated and unmethylated DNA through the use of fluorescently labeled methylation-specific primers. The primers inherently provide both amplification and detection functions, with their fluorescence signals automatically indicating the methylation status without requiring separate detection reactions or additional reagents.
3Measurement precision
If conventional methods use multiple aliquots of test sample for separate measurements, then complete quantification of methylated and unmethylated DNA is achieved, but sample consumption increases
Solution Approach 1:
The patent combines the quantification of both methylated and unmethylated DNA into a single reaction mixture using a small aliquot of the test sample. The methylation-specific primers amplify both DNA forms simultaneously in the same reaction vessel, and the fluorescent signals from the primers allow direct comparison and calculation of the methylation percentage without needing to process separate aliquots.
4Measurement precision
If conventional methods perform multiple separate measurements with external standards, then comprehensive data is obtained, but error propagation increases
Solution Approach 1:
The patent combines the measurement of methylated and unmethylated DNA into a single real-time PCR reaction, which eliminates the propagation of errors between separate measurements. By obtaining both fluorescent signals from one reaction with a single standard curve, the method avoids cumulative errors from multiple standard curve constructions and multiple separate quantification steps.
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 simplifies the measurement of DNA methylation levels by eliminating the need for external standards and reducing errors, providing a more efficient and accurate method for determining the percentage of methylated DNA without the need for extensive DNA handling or multiple reaction mixtures.
Implementation Method 1
providing quantitative amplification data from a sample comprising nucleic acid target, wherein said nucleic acid target comprises at least one copy of a non-variant form of said nucleic acid target and/or at least one copy of a variant form of said nucleic acid target
Implementation Method 2
quantitative amplification data correlates to amplification cycle numbers
Data Source
AI summary
Provided herein is technology relating to evaluating the state of nucleic acids and particularly, but not exclusively, to methods for measuring variations between DNAs, including differences in methylation and mutation.