Double-Stranded Oligonucleotide Probe Reduces Fluorescence Background
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Solution Overview
Problem
Current fluorescence probe methods, such as TaqMan probes, suffer from high fluorescence background and limited sensitivity in gene detection, which hinders accurate qualitative and quantitative analysis and medical diagnosis.
Innovation Solution
A double-stranded oligonucleotide nucleic acid probe is developed, where two partially or completely base-complementary probes are synthesized with fluorescent and quenching groups at their ends, allowing for thorough quenching and reduced background fluorescence, enabling more sensitive detection by separating and binding to PCR products with Taq enzyme activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If TaqMan probe technology is used for fluorescence detection, then specific amplification product detection is achieved, but fluorescence background is high and detection sensitivity is limited
Solution Approach 1:
The probe is divided into two separate oligonucleotide chains (first chain and second chain) that are base-complementary to each other. Each chain is independently labeled with fluorescent and quenching groups. This segmentation allows the probe to form a double-stranded structure that enhances quenching efficiency and reduces background fluorescence while maintaining detection capability.
Solution Approach 2:
The invention uses a composite probe structure consisting of two different oligonucleotide chains with complementary base sequences. The first chain contains fluorescent group F1 and quenching group Q1, while the second chain contains fluorescent group F2 and quenching group Q2. This composite structure enables simultaneous multiple quenching interactions, significantly reducing background fluorescence and improving detection sensitivity.
2Object-generated harmful factors
If single-stranded probe is used, then probe synthesis is simple, but quenching is insufficient and fluorescence background is high
Solution Approach 1:
Two separate oligonucleotide chains are combined to form a double-stranded probe structure through base pairing. The first chain and second chain are complementary to each other, creating a stable double-stranded structure that enhances quenching efficiency. This merging approach reduces fluorescence background while the complementary design simplifies synthesis compared to traditional single-stranded probes requiring multiple modifications.
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 double-stranded probe significantly reduces fluorescence background and enhances detection sensitivity, allowing for accurate quantitative analysis and simultaneous detection of multiple genes with improved specificity and sensitivity.
Implementation Method 1
5′ end of the probe labels a fluorescent molecule, 3′ end labels a corresponding fluorescent quenching molecule
Implementation Method 2
The fluorescence probe method relies on fluorescent resonance energy transfer (FRET) to realize detection
Implementation Method 3
activate the 5′ end exonuclease activity of the Taq enzyme, and cleave the probe into a mononucleotide
Implementation Method 4
the two probes are each labeled with a fluorescent group at 5′ end as a reporter molecule (F1/F2), the two probes are each labeled with a fluorescent quenching molecule (Q1/Q2) at 3′ end corresponding to the 5′ end, and the two probes are completely or partially base-complementary
Data Source
AI summary
Disclosed by the present application are a structure of a double-stranded oligonucleotide nucleic acid probe, a method of use and applications thereof in nucleic acid fluorescence qualitative and quantitative analysis, medical diagnosis and life science researches. The double-stranded oligonucleotide nucleic acid probe is composed of two completely or partially base-complementary oligonucleotide strands; the end of each oligonucleotide strand may be connected to a fluorescent group or a corresponding fluorescent quenching group; and the two oligonucleotide probe strands may hybridize with a target nucleic acid sequence to be tested.