Probe Oligonucleotide Design for Reduced Background Signal in PCR
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Solution Overview
Problem
Current PCR-based detection systems for nucleotide polymorphism (SNP) genotyping lack probes with distinguishable fluorescence characteristics when hybridized and not hybridized to a target nucleic acid sequence, and there is a need for probes with efficient synthesis, high specificity, and proximity-based quenching positioning of reporter and quencher molecules.
Innovation Solution
A method involving tagging the target nucleotide sequence with a nucleotide tag sequence, using a probe oligonucleotide with a nucleotide tag recognition sequence and a regulatory sequence, and amplifying the tagged sequence in a PCR reaction with a probe oligonucleotide and a regulatory oligonucleotide, where the melting temperatures of the oligonucleotides are higher than the annealing temperature, to reduce background signal and enhance assay specificity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional probes are used in PCR-based detection systems, then the detection can be performed, but the background signal is high and specificity is reduced
Solution Approach 1:
The probe is pre-hybridized to the regulatory oligonucleotide before the PCR reaction, forming a complex where the reporter and quencher are in close proximity. This preliminary action ensures that the probe is already in a quenched state before encountering the target sequence, reducing background signal. During PCR, when the probe hybridizes to the target, the regulatory oligonucleotide is displaced, separating the quencher from the reporter and generating a specific signal only when target is present.
Solution Approach 2:
The regulatory oligonucleotide acts as an intermediary that controls the interaction between the probe and the detection system. It binds to the probe in advance and regulates the reporter-quencher proximity. When the target sequence is present during PCR, it competes for the probe, displacing the regulatory oligonucleotide and thereby controlling the fluorescence signal generation, which improves specificity by ensuring signal only occurs with target presence.
2Adaptability or versatility
If multiple probes are synthesized to detect different nucleotide sequences, then detection coverage is improved, but synthesis complexity and cost increase
Solution Approach 1:
The probe design incorporates a universal regulatory oligonucleotide sequence that can be paired with different target-specific recognition sequences. This allows a single probe structure with standardized regulatory elements to be used across multiple detection assays by simply changing the target-specific portion of the probe. The regulatory oligonucleotide serves multiple functions: controlling reporter-quencher proximity, enabling pre-hybridization, and facilitating displacement during PCR, making the system versatile for detecting different nucleotide sequences without redesigning the entire probe architecture.
3Stability of the object's composition
If reporter and quencher molecules are positioned far apart on the probe, then probe stability is improved, but quenching efficiency decreases
Solution Approach 1:
The system uses dynamic control of reporter-quencher proximity through temperature-dependent hybridization and displacement reactions. At lower temperatures during pre-hybridization, the probe forms a stable complex with the regulatory oligonucleotide, bringing reporter and quencher close together for efficient quenching. During the PCR annealing step at higher temperatures, the target sequence competes and displaces the regulatory oligonucleotide, dynamically changing the spatial arrangement. This dynamic behavior allows the same probe structure to achieve both stability and variable quenching efficiency based on reaction conditions.
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 provides a superior assay with reduced background signal and increased specificity, allowing for efficient detection of target nucleotide sequences with few synthesized probes and high sensitivity.
Implementation Method 1
the first label and the second label constitute a fluorescent reporter/quencher pair
Implementation Method 2
a probe oligonucleotide comprising a nucleotide tag recognition sequence that hybridizes to the complement of the nucleotide tag sequence
Implementation Method 3
amplifying the tagged target nucleic acid sequence in a PCR amplification reaction
Data Source
Figure 1
AI summary
The invention provides a method for detecting a target nucleotide sequence by tagging the nucleotide sequence with a nucleotide tag, providing a probe oligonucleotide with a melting temperature Tm1, comprising a regulatory sequence and a nucleotide tag recognition sequence; incorporating the probe oligonucleotide into the tagged polynucleotide in a polynucleotide amplification reaction, providing a regulatory oligonucleotide with a melting temperature Tm2, comprising a sequence segment that is at least partially complementary to the regulatory sequence, amplifying the tagged target nucleic acid sequence in a PCR amplification reaction using the probe oligonucleotide as a primer, and detecting the amplification product; wherein Tm1 and Tm2 are higher than the annealing temperature associated with the polynucleotide amplification reaction.