Double-Functional Oligonucleotide for Real-Time PCR Detection
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Solution Overview
Problem
Current real-time PCR methods face challenges with specificity, sensitivity, and efficiency due to the need for multiple probes and primers, which can lead to non-specific amplification and limited amplification size, especially when detecting large DNA products.
Innovation Solution
A double-functional oligonucleotide (DFO) with an inosine linker and mis-matched nucleotide sequences is used, allowing for dual function as a primer and probe, with a reporter dye and quencher molecule attached, enabling specific gene amplification and fluorescence detection across a wide range of sizes (50 bp to 1000 bp) by controlling annealing temperature.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If multiple probes and primers are used for real-time PCR detection, then measurement precision and reliability are improved, but device complexity and cost increase
Solution Approach 1:
The patent combines the functions of multiple probes and primers into a single double-functional oligonucleotide (DFO) molecule. The DFO contains both a recognition region for specific target binding and a reporter region for fluorescence detection, eliminating the need for separate probes and primers while maintaining detection accuracy
Solution Approach 2:
The DFO oligonucleotide serves multiple functions simultaneously: it acts as a primer for DNA synthesis, a probe for specific target recognition, and a reporter for fluorescence detection. This multi-functional design reduces the number of components needed in the real-time PCR system
2Productivity
If standard PCR methods are used for amplifying large DNA products, then amplification capability is maintained, but productivity and efficiency decrease
Solution Approach 1:
The patent modifies the oligonucleotide parameters by creating a double-functional structure with specific length ranges (recognition region: 15-30 nucleotides, reporter region: 10-20 nucleotides) and controlled secondary structures that enhance amplification efficiency for large DNA products while maintaining the ability to amplify various sizes from 50 bp to 1000 bp
3Reliability
If conventional probe designs are used, then specificity is achieved, but device complexity and cost increase
Solution Approach 1:
The patent merges the specificity-providing recognition region with the detection-providing reporter region into a single DFO oligonucleotide molecule, reducing the number of separate oligonucleotides from multiple probes and primers to just one double-functional molecule while maintaining high specificity
Solution Approach 2:
The DFO oligonucleotide universally performs multiple functions: specific target recognition through its recognition region, DNA synthesis initiation as a primer, and fluorescence detection through its reporter region, all within a single molecule
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 DFO achieves high specificity and sensitivity equivalent to Taqman systems using only two oligonucleotides, allowing for real-time detection of large DNA products with reduced complexity and cost, while maintaining efficiency across various amplification sizes.
Implementation Method 1
the fluorescent substance, reporter, and extinction material, quencher, as a pair in the nucleotide sequence complementary to the 5′-terminus of this primer
Data Source
AI summary
The present disclosure relates to a complementary double-stranded oligo, in which, for the amplification of a particular gene sequence, an inosine linker is linked to the 5′-terminus of a primer for the corresponding sequence, a sequence complementary to the primer is linked to the inosine linker, and at least one mis-matched nucleotide is included in the complementary sequence to form a bubble structure; in which, depending on the treatment temperature, at a predetermined temperature or lower, a single stranded oligo is turned into a double-stranded form to exist in an inactivation form, and at a predetermined temperature or higher, the oligo is activated into a single-stranded oligo; and in which, a fluorescent substance (reporter dye) and a quenching material (quencher molecule) are attached to the oligo, so that the oligo can be applied as a primer or a probe, and thus only two oligos can realize the gene amplification and fluorescent signal real-time measurement with high specificity, and to a measuring method after a fluorescent arrangement step using the oligomer is added. The present disclosure has advantages in view of an oligo and a design method therefor, in which an oligo capable of simultaneously performing amplification and detection with an amplification size of 500 bp to 1000 bp for a particular gene is used to allow a reporter dye and a quencher molecule to be attached to only two target gene amplification sites, thereby confirming whether real-time amplification occurs.