Labeled Primer Set for Nucleic Acid Mutation Detection
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Solution Overview
Problem
Conventional fluorescent substances used for detecting target nucleic acid sequences have limitations, such as false fluorescence emission in the absence of a double helix structure and high costs due to the need for two types of dyes, and existing methods like thiazole orange suffer from low sensitivity due to strong fluorescence with single-stranded DNA, making effective detection of double helix formation challenging.
Innovation Solution
A labeled primer set with specific structures represented by formulae (16), (16b), (17), and (17b) is used for amplifying target nucleic acid sequences, incorporating fluorescent and exciton-effect groups that enhance fluorescence only upon double helix formation, allowing for sensitive detection of nucleic acid amplification and mutations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional fluorescent substances are used for detecting target nucleic acid sequences, then fluorescence emission occurs, but false fluorescence emission occurs even when no double helix structure has been formed
Solution Approach 1:
The patent introduces a specific fluorescent substance as an intermediary that requires double helix formation as a mediator to activate fluorescence. The fluorescent substance itself does not emit fluorescence directly but only when incorporated into a double helix structure, thus using the double helix as a necessary mediator between the fluorescent substance and the detection signal.
Solution Approach 2:
The patent changes the fluorescence emission parameter of the detection system by using a fluorescent substance whose fluorescence intensity is contingent upon the formation of a double helix structure. This parameter change ensures that fluorescence is only emitted under the specific condition of double helix formation, eliminating false positives.
2Illumination intensity
If thiazole orange is used as a fluorescent dye, then strong fluorescence is emitted through interaction with single-stranded DNA, but the increase in fluorescence intensity when double helix is formed is small
Solution Approach 1:
Instead of using a fluorescent dye that emits strong fluorescence with single-stranded DNA and shows only a small increase with double helix formation, the patent inverts this approach by using a fluorescent substance that shows minimal fluorescence in single-stranded form but exhibits strong fluorescence enhancement specifically when double helix structure is formed.
3Measurement precision
If fluorescence resonance energy transfer (FRET) is used to quench fluorescence of primer or probe, then fluorescence quenching is achieved, but cost increases due to introduction of two types of fluorescent dyes
Solution Approach 1:
The patent extracts the need for a separate quenching mechanism by using a single fluorescent substance that inherently provides both the fluorescent signal and the quenching function through its interaction with the double helix structure. This eliminates the requirement for a second fluorescent dye or quenching molecule, thereby reducing cost.
Solution Approach 2:
The fluorescent substance used in the patent serves multiple functions: it acts as the fluorescent signal source and simultaneously provides the quenching mechanism through its specific interaction with double helix structures. This multi-functionality eliminates the need for separate components and reduces overall cost.
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
The labeled primer set enables effective detection of nucleic acid amplification and mutation presence with high sensitivity, suitable for various applications including clinical diagnostics and gene detection, by utilizing fluorescence changes associated with double helix formation, thus improving detection specificity and efficiency.
Implementation Method 1
a fluorescent substance whose fluorescence intensity increases with an increase in target nucleic acid sequence. A typical example of the fluorescent substance is a substance that intercalates into a double helix structure and emits fluorescence by irradiation with excitation light
Implementation Method 2
a substance that intercalates into a double helix structure and emits fluorescence by irradiation with excitation light
Data Source
Figure 1(A)~1(B)
Figure 2
Figure 3
AI summary
The present invention provides a primer that effectively can detect, for example, the double helix structure of a nucleic acid. The primer is a labeled nucleic acid containing at least one structure represented by the following formula (16), where B is an atomic group having a nucleobase skeleton, E is an atomic group having a deoxyribose skeleton, a ribose skeleton, or a structure derived from either one of them, or an atomic group having a peptide structure or a peptoid structure, and Z11 and Z12 are each an atomic group that exhibits fluorescence and are identical to or different from each other.