Oligonucleotide Detection Probes for Nucleic Acid Sequences
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Solution Overview
Problem
Current methods for detecting nucleic acid molecules, particularly for diagnosing diseases through gene expression analysis, lack sensitivity and specificity, and are not rapid enough to effectively identify mutations or gene signatures associated with disorders.
Innovation Solution
A method involving oligonucleotide detection probes and bifunctional oligonucleotides that specifically bind to target nucleic acid sequences, using an anchor immobilized on a surface to tether the nucleic acid molecules, allowing for ligation and subsequent detection of the presence or amount of the target nucleic acid, even in the presence of nucleotide variants.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional detection methods are used, then the detection process is simpler, but the sensitivity and specificity are insufficient
Solution Approach 1:
The detection probe is divided into two functional segments: a target-binding portion that specifically binds to the nucleic acid target and an anchor-binding portion that binds to a solid support anchor. This segmentation allows the probe to perform both target recognition and stable immobilization functions separately, enhancing detection sensitivity while maintaining manageable complexity
Solution Approach 2:
The invention transitions from solution-phase detection to surface-immobilized detection by anchoring the probe to a solid support. This dimensional change from 3D solution environment to 2D surface configuration increases local probe concentration, improves signal-to-noise ratio, and enables washing away unbound materials, thereby significantly enhancing detection specificity
2Measurement precision
If the detection method is made more sensitive, then variant detection improves, but the detection time increases
Solution Approach 1:
The probe is pre-immobilized on the solid support before sample addition, and the target-binding portion is pre-configured with optimal affinity characteristics. This preliminary preparation allows rapid binding of target nucleic acids upon sample addition, reducing detection time while maintaining high sensitivity for variant detection
Solution Approach 2:
The invention optimizes the binding parameters of the probe by adjusting the length and sequence composition of the target-binding portion to achieve high affinity for specific variants. This parameter optimization enables rapid and specific binding at physiological temperatures, simultaneously improving detection speed and accuracy
3Measurement precision
If specific binding conditions are used, then detection specificity improves, but the detection speed decreases
Solution Approach 1:
The solid support anchor acts as an intermediary that stabilizes the probe in a conformation optimized for specific target binding. The anchor-probe-target complex formation provides multiple binding interfaces simultaneously, allowing stringent specificity requirements to be met without sacrificing binding kinetics
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 sensitive and specific detection of nucleic acid molecules, including variants, by ensuring that only perfectly complementary probes are ligated and remain bound to the surface, while mismatches are removed, thereby accurately quantifying target nucleic acids in a sample.
Implementation Method 1
contacting the sample with at least one oligonucleotide detection probe that specifically binds to a first target sequence of the target nucleic acid molecule
Implementation Method 2
at least one bifunctional oligonucleotide including a portion that specifically binds to a second target sequence of the target nucleic acid molecule and a portion that specifically binds to an anchor
Implementation Method 3
adding a ligase to the tethered detection probe and the tethered bifunctional oligonucleotide, thereby producing a ligated detection probe and bifunctional oligonucleotide
Implementation Method 4
adding a reagent to specifically remove substantially all tethered target nucleic acid molecule, wherein the reagent has substantially no specific activity to remove the ligated detection probe and bifunctional oligonucleotide
Data Source
Figure 1A~1D
Figure 2A~2C
Figure 3A~3B
AI summary
Disclosed herein are methods for detecting a target nucleic acid molecule in a sample. The methods can include contacting a sample with a detectably labeled probe (detection probe) that specifically binds to a first target sequence in the target nucleic acid molecule, a bifunctional oligonucleotide including a portion that specifically binds to a second target sequence in the target nucleic acid molecule and a portion that specifically binds to an anchor, and a surface comprising the anchor. Specifically bound detection probe and bifunctional oligonucleotide are ligated and a reagent that specifically removes substantially all of the target nucleic acid is added. Unligated detection probe is removed and presence of the detectable label is detected. In other embodiments, the ligation and/or removal of target nucleic acid are omitted and the detection probe specifically bound to the target nucleic acid is detected.