Asymmetric DNA Amplification Primer for Beacon Binding
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Solution Overview
Problem
In asymmetric nucleic acid amplification, one strand of the DNA excessively amplified forms an intramolecular secondary structure, reducing the binding efficiency of molecular beacons and leading to lowered fluorescence intensity, which can make DNA detection in digital PCR challenging due to undetectable fluorescence and impaired melting curve analysis.
Innovation Solution
Introducing a mutation into the primer sequence used in asymmetric nucleic acid amplification to increase the proportion of single-strand forming bases in the probe binding region of the amplified DNA, thereby enhancing the binding efficiency of molecular beacons and improving fluorescence intensity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If asymmetric nucleic acid amplification is performed to excessively amplify one strand complementary to the molecular beacon, then the binding of the molecular beacon with the target DNA is enhanced, but the excessively amplified DNA forms an intramolecular secondary structure that reduces the binding efficiency and lowers fluorescence intensity
Solution Approach 1:
The patent modifies the primer sequence by introducing mutations that change the secondary structure of the amplified DNA. Specifically, the primer sequence is designed to prevent the formation of intramolecular secondary structures in the probe binding region, thereby changing the structural parameters of the amplified DNA from double-stranded to single-stranded conformation, which improves molecular beacon binding efficiency and restores fluorescence intensity
Solution Approach 2:
The patent extracts or removes the problematic secondary structure formation from the amplified DNA by designing primers that prevent complementary base pairing in the probe binding region. This eliminates the harmful intramolecular structure that was causing reduced binding efficiency, allowing the molecular beacon to access its target sequence effectively
2Stability of the object's composition
If the DNA forms a double-stranded structure in the probe binding region, then the amplified DNA is stable, but the molecular beacon cannot bind effectively, leading to undetectable fluorescence in digital PCR
Solution Approach 1:
The patent changes the secondary structure parameter of the amplified DNA by introducing mutations in the primer sequence. These mutations prevent the formation of stable double-stranded structures in the probe binding region, converting it to a single-stranded conformation that is accessible to the molecular beacon, thereby enabling fluorescence detection while maintaining overall DNA stability through proper primer design
3Productivity
If the intramolecular secondary structure is formed in the excessively amplified strand, then the asymmetric amplification is efficient, but the melting curve analysis becomes difficult due to too small change in fluorescence intensity
Solution Approach 1:
The patent modifies the secondary structure parameters of the amplified DNA by introducing mutations that eliminate intramolecular pairing in the probe binding region. This structural change ensures that the DNA remains in a single-stranded conformation suitable for molecular beacon binding, thereby producing sufficient fluorescence signal change during melting curve analysis to enable accurate genotyping while maintaining asymmetric amplification efficiency
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 sensitive detection of target genes by increasing the proportion of single-strand structures, which enhances the binding of molecular beacons and improves the sensitivity and accuracy of genotyping.
Implementation Method 1
a molecular beacon, which can form a stem-loop in a free state and can bind with a target DNA to be detected at the loop part
Implementation Method 2
use an intercalator or a fluorescent-labeled probe to detect DNA
Data Source
Figure 1A~1C
Figure 2A~2B
Figure 3A~3C
AI summary
The present invention relates to a primer, a DNA detection method, and a DNA detection kit for use in nucleic acid amplification, and particularly in asymmetric nucleic acid amplification. More specifically, this invention relates to a primer for introducing a mutation into a test nucleic acid and amplifying the test nucleic acid, the primer contains, in its sequence, a mutation to be introduced into the test nucleic acid, so as to increase a proportion of single-strand forming bases in a probe binding region of the nucleic acid amplified from the test nucleic acid, at a temperature of the binding of the nucleic acid amplified from the test nucleic acid with the probe; and, a DNA detection method and a DNA detection kit with use of such primer.