Nucleic Acid Probe Cleavage for Multiplex Detection
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Solution Overview
Problem
Current nucleic acid detection methods, such as homogeneous assays, are limited in their ability to perform multiplex detection due to the need for different dyes for each target nucleic acid, and existing solutions using cleavable tags face challenges in accurately correlating detection tags with target nucleic acids, hindering high-throughput multiplex detection.
Innovation Solution
A method involving oligonucleotide probes with a non-complementary 'flap' region attached to the 5′-end, which generates a unique cleavage fragment during PCR, allowing for identification by mass spectroscopy, enabling rapid high-throughput multiplex detection of nucleic acid targets by producing fragments with unique and resolvable masses.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If homogeneous assays use different dyes for each target nucleic acid, then detection of multiple targets is possible, but the complexity of the assay increases and multiplexing capability is limited
Solution Approach 1:
The invention uses a universal detection method (mass spectrometry) that can detect multiple target nucleic acids simultaneously without requiring different dyes or labels for each target. The mass spectrometer serves as a universal detector that distinguishes targets based on the unique mass of cleaved probe fragments, enabling multiplexing while maintaining assay simplicity.
Solution Approach 2:
The invention changes the detection parameter from optical properties (dye fluorescence) to mass properties (fragment mass). By designing probes with unique mass-distinguishable fragments that are cleaved during PCR amplification, the system enables multiplex detection through mass spectrometry, where each target produces a characteristic mass signature rather than requiring a unique dye.
2Measurement precision
If cleavable tags are used in oligonucleotide probes, then detection of target nucleic acid is enabled, but accurate correlation of detection tags with target nucleic acid remains problematic
Solution Approach 1:
The probe is segmented into distinct functional portions: a target-complementary region that anneals to the target nucleic acid and a non-complementary tag portion that is cleaved off. This segmentation ensures that only the tag portion is released and detected, providing clear correlation between probe binding and tag detection without interference from unbound probes.
Solution Approach 2:
The invention uses the PCR amplification process as an intermediary mechanism that links target detection to probe cleavage. The DNA polymerase's 5' nuclease activity mediates the cleavage of the probe tag during amplification, creating a reliable causal chain: target presence → probe annealing → polymerase-mediated cleavage → tag release → detection, thereby ensuring accurate correlation.
3Productivity
If multiple dyes are used for each target nucleic acid, then each target can be detected specifically, but the throughput and efficiency of detection are reduced
Solution Approach 1:
A single mass spectrometer detector performs the function of multiple dye-based detection systems. The instrument can simultaneously detect and distinguish multiple target nucleic acids based on the unique mass of each probe's cleaved fragment, eliminating the need for multiple dyes while maintaining the ability to detect multiple targets in parallel.
Solution Approach 2:
The invention uses the natural PCR amplification process to generate multiple copies of the target nucleic acid and corresponding probe fragments. This amplification creates sufficient signal for detection without requiring multiple different detection reagents, as the mass spectrometer detects the accumulated cleaved fragments from amplified products.
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 enables accurate and efficient detection of multiple nucleic acid targets by generating unique mass-distinguishable fragments, facilitating high-throughput multiplex detection without the need for multiple dyes, thereby overcoming the limitations of existing methods.
Implementation Method 1
the 5′ to 3′ nuclease activity of said nucleic acid polymerase is able to cleave and release from the annealed oligonucleotide probe, fragments containing the second portion of the oligonucleotide probe
Implementation Method 2
treating said fragments containing the second portion of the oligonucleotide probe with a 3′ to 5′ exonuclease that cleaves said fragments up to the exonuclease-resistant modification thereby producing a single fragment having a unique mass-distinguishable size
Implementation Method 3
subjecting the reaction mixture to an affinity matrix that recognizes and binds to the affinity label on said pair of oligonucleotide primers and on said oligonucleotide probe, thereby removing excess oligonucleotide primers and uncleaved oligonucleotide probes
Implementation Method 4
detecting the presence or absence of the single fragment by mass spectrometry, thereby detecting the presence or absence of the target nucleic acid sequence in the sample
Data Source
AI summary
The present invention provides for novel methods and compositions for nucleic acid sequence detection. Unique, identifying cleavage fragments from probes, bound to target nucleic acids, are produced during PCR by the 5′-nuclease activity of the polymerase. The identity of the targets can be determined by identifying the unique cleavage fragments.