Melting Peak Analysis for Nucleic Acid Quantification
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Solution Overview
Problem
Conventional methods for quantifying target nucleic acid sequences using melting analysis are limited by variability in results due to amplification cycle termination and require longer analysis times, especially when detecting multiple sequences simultaneously.
Innovation Solution
A method that involves amplifying target nucleic acid sequences to form a duplex with a labeling moiety, performing melting analysis at two to five predetermined cycles, and using the melting peak curve to quantify the sequence, allowing for simultaneous detection and quantification of multiple sequences in a more rapid and accurate manner.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional melting analysis is performed after complete amplification, then target detection is achieved, but quantification accuracy deteriorates due to variability from amplification cycle termination
Solution Approach 1:
The patent performs melting analysis at predetermined cycles (2-5 cycles) during the amplification process rather than after complete amplification. This preliminary action at standardized time points eliminates variability caused by different amplification termination points, thereby improving quantification accuracy and result reliability.
2Measurement precision
If melting analysis is performed at every amplification cycle to ensure accurate quantification, then measurement precision improves, but analysis time increases significantly
Solution Approach 1:
The patent extracts and performs melting analysis only at specific predetermined cycles (2-5 cycles) during amplification, rather than analyzing every cycle. This selective approach maintains sufficient quantification accuracy while significantly reducing the total analysis time required.
3Adaptability or versatility
If multiple fluorescent molecules are used to detect multiple target sequences simultaneously, then detection capability improves, but spectrum interference increases
Solution Approach 1:
The patent replaces fluorescent label-based detection with melting temperature-based detection. Since melting temperature is an intrinsic physical property of the DNA duplex independent of fluorescent labels, this substitution eliminates spectrum interference while maintaining multiplex detection capability through differential melting temperatures of different target sequences.
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 provides more accurate and rapid quantification of target nucleic acid sequences by utilizing the melting peak curve data from specific cycles, overcoming the limitations of conventional methods and enabling simultaneous detection of multiple sequences with a single detecting channel.
Implementation Method 1
performing a melting analysis at two to five predetermined cycles during the repetition in the step (a) by measuring a melting peak curve obtained by melting the duplex
Data Source
AI summary
The present invention relates to a method for quantifying a target nucleic acid sequence performed in such a manner that at least two cycles in the nucleic acid amplification subject to melting peak analysis are predetermined before the nucleic acid amplification and melting peak analyses are performed for the at least two predetermined cycles, followed by quantifying the target nucleic acid sequence using data values from the melting peak curve (e.g., the presence or absence, height and area).