Cationic Oligonucleotide Conjugates for Low-Background PCR Probes
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Solution Overview
Problem
Existing real-time fluorescence quantitative PCR methods face challenges in probe design length versus melting temperature (TM) balance, leading to non-specific amplification and reduced sensitivity due to electrostatic repulsion and probe length limitations.
Innovation Solution
An oligonucleotide conjugate with a cationic unit is developed, where the cationic unit is coupled to the quenching group, enhancing probe stability and specificity, allowing for flexible probe design and improved sensitivity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If the probe length is shortened to increase discrimination for single nucleotide genotyping and provide design flexibility, then the melting temperature (TM value) decreases, which inhibits the effective binding of the probe to the target sequence at the PCR cycle temperature
Solution Approach 1:
The patent introduces a cationic unit to modify the electrostatic properties of the probe, changing the physical-chemical parameters to enhance binding affinity. This allows shorter probes to maintain effective binding by compensating for the reduced length through enhanced electrostatic interaction with the target sequence.
Solution Approach 2:
The patent creates a composite structure by coupling a cationic unit with the oligonucleotide probe. This composite material combines the sequence-specific binding capability of the oligonucleotide with the electrostatic enhancement provided by the cationic unit, achieving both short probe length and high binding effectiveness.
2Device complexity
If conventional probes are used without cationic units, then the probe structure is simple, but non-specific amplification occurs and sensitivity is reduced due to electrostatic repulsion
Solution Approach 1:
The patent converts the potentially harmful electrostatic repulsion between negative phosphate groups into a beneficial interaction by introducing a cationic unit. The cationic unit creates electrostatic attraction that enhances specific binding while the quenching group maintains the probe's ability to distinguish specific sequences, thereby improving sensitivity without excessive complexity.
3Ease of manufacture
If the probe is designed with standard structures, then the manufacturing process is simple, but fluorescence background is high and sensitivity is reduced
Solution Approach 1:
The patent introduces a quenching group as an intermediary element between the fluorescent reporter and the target sequence. This quenching group absorbs excess fluorescence energy, reducing background signal while allowing the fluorescent reporter to maintain its ability to signal specific binding events, thereby improving signal clarity without complicating the manufacturing process.
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 oligonucleotide conjugate improves probe specificity, reduces fluorescence background, and enhances sensitivity, while allowing for adjustable TM values and increased amplification efficiency.
Implementation Method 1
the cationic unit is coupled to the quenching group, enhancing probe stability and specificity
Implementation Method 2
a reporter fluorescent group and a quenching group are labeled at both ends of the sequence respectively
Implementation Method 3
During PCR extension, the heat-resistant Taq DNA polymerase degrades the probe with its 5'-3' exonuclease activity to separate the reporter fluorescent group and the quenching fluorescent group
Implementation Method 4
At the annealing temperature, the probe begins to bind to one of the single strands of DNA
Data Source
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AI summary
The present invention provides an oligonucleotide conjugate with high hybridization performance and use thereof. The oligonucleotide conjugate of the present invention can be used as a probe for nucleic acid detection, the oligonucleotide conjugate of the present invention can be used to design probes more flexibly, and the conservative regions are more accessible. The specificity of the probe is better, and the fluorescence value of the probe can be significantly improved, the fluorescence background is significantly reduced, and the sensitivity of the probe is greatly improved.