FRET-Labeled Oligonucleotide Detection in Amplification Reactions
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current nucleic acid analyte detection methods, particularly in biomedical applications, require secondary techniques like gel electrophoresis or mass spectroscopy for analyte detection, which are inefficient, time-consuming, and costly.
Innovation Solution
The use of oligonucleotides attached to FRET chromophores in amplification reactions to directly detect polynucleotides through FRET signals, allowing for the detection of analytes by measuring energy transfer between FRET donor and acceptor chromophores during polymerization reactions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If amplification-based detection technology is used with secondary techniques (gel electrophoresis, mass spectroscopy), then analyte detection can be achieved, but detection efficiency is low and time consumption increases
Solution Approach 1:
The patent combines amplification and detection into a single integrated reaction system. FRET-labeled probes are incorporated during the amplification process, allowing real-time monitoring of analyte detection without requiring separate secondary techniques like gel electrophoresis or mass spectroscopy. This merging of functions directly resolves the contradiction by eliminating the time-consuming separation steps while maintaining detection capability.
Solution Approach 2:
The patent replaces mechanical separation methods (gel electrophoresis) and complex analytical instruments (mass spectroscopy) with a optical detection system based on FRET signaling. The mechanical/physical separation processes are substituted by a biochemical energy transfer mechanism that provides direct, real-time detection during amplification, thereby improving productivity and reducing time loss.
2Productivity
If amplification-based detection technology is used with secondary techniques, then analyte detection can be achieved, but cost increases
Solution Approach 1:
By merging amplification and detection into one reaction system using FRET-labeled probes, the patent eliminates the need for expensive secondary techniques such as gel electrophoresis equipment and mass spectroscopy instruments. This integration reduces overall system cost while maintaining high detection efficiency through real-time optical monitoring.
Solution Approach 2:
The patent substitutes expensive mechanical and instrumental analysis methods with a cost-effective optical detection approach. FRET-based fluorescence measurement uses standard fluorescent microscopy or flow cytometry equipment, replacing the need for costly gel electrophoresis gels, staining reagents, and mass spectroscopy instruments, thereby reducing manufacturing and operational costs.
3Productivity
If FRET chromophores are attached to oligonucleotides for direct detection, then detection efficiency improves, but device complexity increases
Solution Approach 1:
The patent uses FRET chromophores as intermediary molecules that bridge the amplification process and detection signal. The FRET pair (donor and acceptor chromophores) acts as a mediator that translates biochemical amplification events into optical signals, enabling direct detection without complex instrumentation. This intermediary approach improves detection efficiency while keeping the overall system relatively simple.
Solution Approach 2:
The patent exploits changes in optical parameters (fluorescence intensity ratios) of the FRET chromophores to detect analyte presence. By monitoring the FRET signal intensity and spectral characteristics, the system converts complex biochemical information into simple optical parameter changes that can be detected with standard instruments, thereby improving detection efficiency without proportionally increasing system complexity.
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 method enables direct and efficient detection of polynucleotides and genetic variations, reducing the need for secondary techniques and improving detection efficiency and cost-effectiveness.
Implementation Method 1
The first FRET chromophore and the second FRET chromophore are capable to provide an energy transfer from one to another when located at a Förster distance one with respect to the another, thus forming a FRET donor-acceptor chromophores pair
Data Source
AI summary
FRET-based analytes detection and related methods and systems are described where a pair of FRET labeled primers and/or oligonucleotides are used that are specific for target sequences located at a distance up to four time the Förster distance of the FRET chromophores presented on the FRET labeled primers and/or oligonucleotides one with respect to the other in one or more polynucleotide analyte; in particular the pair of FRET labeled primers and/or oligonucleotides is combined with a sample and subjected to one or more polynucleotide amplification reactions before measuring FRET signals from at least one FRET chromophore.