Base Sequence Analysis Apparatus Turbidity Detection
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Solution Overview
Problem
Current methods for base sequence analysis, such as melting curve analysis, require a separate switch from nucleic acid amplification to analysis, which can be inefficient and may hinder the accuracy of the analysis due to the presence of probe nucleic acids during amplification.
Innovation Solution
A method and apparatus that enable consecutive nucleic acid amplification and melting curve analysis by incorporating a turbidity measurement procedure to determine when to switch from amplification to analysis mode, using a probe nucleic acid with a lower melting temperature than the amplification reaction, and employing dual light sources for turbidity and fluorescence detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If a separate switch from nucleic acid amplification to melting curve analysis is performed, then the analysis can be carried out, but the efficiency is reduced and accuracy is hindered due to probe nucleic acid interference during amplification
Solution Approach 1:
The patent combines the nucleic acid amplification reaction and melting curve analysis procedures into a single continuous process without intermediate switching. The probe nucleic acid is designed with a melting temperature lower than the amplification reaction temperature, allowing it to remain single-stranded during amplification (avoiding interference) and automatically form double-stranded structures during the subsequent melting curve analysis phase, thereby achieving both high efficiency and high accuracy
Solution Approach 2:
The invention utilizes temperature parameter changes to control the state of the probe nucleic acid. By setting the probe melting temperature below the amplification temperature, the probe remains in a single-stranded state during amplification (non-interfering). When the temperature is lowered for melting curve analysis, the probe automatically forms double-stranded structures, enabling accurate detection without requiring separate switching procedures
2Productivity
If probe nucleic acid is present during amplification reaction, then melting curve analysis can be performed consecutively, but the probe may interfere with the amplification process
Solution Approach 1:
The patent solves this contradiction by controlling the temperature parameter. The probe nucleic acid is designed with a melting temperature (Tm) lower than the amplification reaction temperature. During amplification, the high temperature keeps the probe in a single-stranded state, preventing it from interfering with the amplification process. When the temperature is lowered for melting curve analysis, the probe automatically forms double-stranded structures, enabling accurate detection
Solution Approach 2:
The invention makes the probe nucleic acid dynamic in its structural state based on temperature conditions. The probe transitions from a single-stranded state during amplification (non-interfering) to a double-stranded state during melting curve analysis (detectable), allowing the same probe to serve different functions at different stages without compromising either amplification reliability or analysis capability
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 allows for more accurate and efficient base sequence analysis by ensuring the probe nucleic acid does not interfere with amplification and enables simultaneous detection of amplified products, improving the precision of melting curve analysis.
Implementation Method 1
change from a double strand to a single strand can be detected using an intercalator that produces fluorescent light by intercalating into a double-stranded nucleic acid
Implementation Method 2
dissociation of the nucleic acid chain can be detected based on the emission or extinguishing of light by the fluorescent label when the target nucleic acid chain and the nucleic acid probe dissociate
Implementation Method 3
pyrophosphoric acid produced as a byproduct binds with magnesium ions in the reaction solution, thereby forming magnesium pyrophosphate. If the amount of produced magnesium pyrophosphate exceeds the soluble level in the sample, magnesium pyrophosphate precipitates, and the reaction solution turns cloudy
Data Source
AI summary
There is provided a base sequence analysis method including: a nucleic acid amplification procedure of obtaining an amplification product by a nucleic acid amplification reaction, a turbidity measurement procedure of measuring turbidity of a reaction solution of the nucleic acid amplification reaction; and a melting curve analysis procedure of performing melting curve analysis of a probe nucleic acid chain and the amplification product at a reaction site of the nucleic acid amplification reaction. This base sequence analysis method makes it possible to consecutively perform a nucleic acid amplification reaction and melting curve analysis at the same reaction site.