Single-Labelled Oligonucleotide FRET Detection System
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Solution Overview
Problem
Current FRET-based detection systems for PCR are costly, time-consuming, and prone to non-specific signals, particularly due to the need for dual-labelled oligonucleotide probes and complex synthesis processes, which limits their accessibility and reliability for SNP genotyping and quantitative gene expression analysis.
Innovation Solution
A detection system utilizing single-labelled oligonucleotide sequences with differing melting temperatures (Tm) that hybridize to form a fluorescent quenched pair, where one sequence has a Tm below the PCR annealing temperature, allowing for easier synthesis and reduced costs, and enabling specific signal generation upon introduction of a complementary sequence.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If dual-labelled oligonucleotide probes are used for FRET-based PCR detection, then detection sensitivity and specificity are improved, but production costs and synthesis complexity increase significantly
Solution Approach 1:
The dual-labelled probe is segmented into two separate single-labelled oligonucleotide sequences. One sequence contains the fluorophore label while the other contains the quencher label. These separate sequences hybridize to each other to form the FRET-quenched pair, eliminating the need for expensive dual-labelled probe synthesis while maintaining detection functionality.
Solution Approach 2:
A complementary sequence acts as an intermediary that displaces the single-labelled sequences from each other. When the complementary sequence is introduced, it binds to one of the single-labelled sequences, causing a conformational change that separates the fluorophore from the quencher and generates the fluorescent signal.
2Measurement precision
If dual-labelled oligonucleotide probes are used for FRET-based PCR detection, then detection specificity is improved, but synthesis time and process complexity increase
Solution Approach 1:
The synthesis process is segmented into two independent steps: first synthesizing the fluorophore-labelled sequence, then synthesizing the quencher-labelled sequence separately. This avoids the complex simultaneous synthesis of dual-labelled probes and allows each sequence to be optimized independently for faster production.
Solution Approach 2:
The single-labelled sequences are synthesized and prepared in advance before the actual detection assay. This preliminary preparation simplifies the overall process by eliminating the need for complex dual-labelled probe synthesis at the time of experimentation, reducing both synthesis time and process complexity.
3Productivity
If conventional FRET-based assays are used, then PCR product detection is achieved, but non-specific signals reduce reliability
Solution Approach 1:
The assay incorporates local quality control through the use of specifically designed single-labelled sequences with defined Tm values. The fluorophore-labelled sequence and quencher-labelled sequence have different Tm characteristics that allow them to hybridize specifically under controlled conditions, reducing non-specific binding and improving signal reliability.
Solution Approach 2:
The assay optimizes detection reliability by controlling the temperature parameter. The single-labelled sequences are designed with Tm values that allow specific hybridization at the assay temperature. By carefully selecting and adjusting temperature parameters, the assay minimizes non-specific signals while maintaining detection capability.
4Ease of manufacture
If single-labelled oligonucleotide sequences with different Tm values are used, then synthesis cost and complexity are reduced, but maintaining detection reliability becomes challenging
Solution Approach 1:
The invention addresses reliability concerns by optimizing the Tm parameter difference between the two single-labelled sequences. By carefully selecting sequences with appropriate Tm values and controlling the assay temperature, the system ensures specific hybridization and reliable detection while maintaining the cost advantages of single-labelled synthesis.
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 significantly reduces production costs and simplifies the synthesis of FRET-based assays, enhancing their reliability and specificity for PCR product detection in both real-time and end-point analyses, while minimizing non-specific signals.
Implementation Method 1
The principle of all the homogeneous assay systems is to use the physical process of Fluorescence Resonance Energy Transfer (FRET) to detect the production of product in the PCR process. FRET is the process whereby when two fluorophores are in close enough proximity to each other that they will undergo an energy transfer exchange when excited by light at wavelengths matched to their particular excitation wavelength.
Implementation Method 2
at least two single-labelled oligonucleotide sequences of differing Tm that hybridise to one another in free solution to form a fluorescent quenched pair, that upon introduction of a complementary sequence to one or both sequences generates a measurable signal, one of the sequences being of a Tm that is below the Ta of the PCR process
Data Source
AI summary
The present invention provides a detection system for a PCR process using FRET which comprises at least two single-labelled Oligonucleotide sequences of differing Tm that hybridize to one another in free solution to form a fluorescent quenched pair, that upon introduction of a complementary sequence to one or both sequences generates a measurable signal, one of the sequences being of a Tm that is below the Ta of the PCR process, the other not being below the Ta of the PCR process.