Oligonucleotide Probe Segmentation for Multiplex Assay Signal Purity
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current multiplex genetic analysis systems face challenges with signal-to-noise ratio degradation at low signal intensity due to residual label or chemical reagent carry-over, leading to unreliable test results and increased intra-sample variability.
Innovation Solution
The method involves using labeled oligonucleotides with a specific structure that allows for hydrolytic separation of the labeled portion during amplification reactions using a DNA polymerase with 5′->3′-exonuclease activity, preventing carry-over and enhancing signal specificity by hybridizing pooled oligonucleotides to a solid phase for deconvolution-based analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If multiple patient samples are analyzed using conventional multiplex systems, then throughput is improved, but signal-to-noise ratio deteriorates due to residual label carry-over
Solution Approach 1:
The oligonucleotide probe is divided into distinct functional segments: a first portion for hybridization to the target nucleic acid, a second portion containing the unique identifier for sample identification, and a labeled portion with the detectable label. This segmentation allows the labeled portion to be selectively removed after hybridization, preventing carry-over interference while preserving the diagnostic signal.
Solution Approach 2:
The labeled portion is extracted from the oligonucleotide probe after hybridization to the solid phase. This extraction removes the source of background signal (residual labels) from the system, eliminating the carry-over problem that degrades signal-to-noise ratio in conventional multiplex systems while maintaining the diagnostic capability of the bound probe portions.
2Productivity
If residual labeled nucleotides are present from amplification reactions, then amplification efficiency is maintained, but background signal increases leading to unreliable test results
Solution Approach 1:
A unique identifier sequence acts as an intermediary between the target nucleic acid and the detectable label. This intermediary allows the label to be attached to the probe for efficient amplification and detection, but enables selective removal of the labeled portion after hybridization, thereby eliminating background signal while preserving amplification efficiency.
Solution Approach 2:
Different portions of the oligonucleotide probe have different fates: the first portion (hybridization region) remains bound to the target on the solid phase to provide the diagnostic signal, while the labeled portion is selectively removed to eliminate background interference. This local differentiation in fate resolves the contradiction between maintaining label presence for detection and removing it for reliability.
3Productivity
If high density arrays are used to increase analysis capacity, then productivity is improved, but signal specificity deteriorates due to carry-over of labeling reagents
Solution Approach 1:
The labeled portion is extracted from the system after hybridization to the high density array. This extraction removes the source of non-specific background signals that would otherwise interfere with the high throughput analysis, allowing the benefits of high density arrays to be fully realized without the penalty of reduced signal specificity.
Solution Approach 2:
The probe structure is segmented into functional regions with different roles: the first portion provides specific hybridization to target sequences on the array, the second portion provides unique identification for sample tracking, and the labeled portion provides detectable signal. This segmentation enables selective removal of the labeled portion while maintaining the specificity-providing portions, thereby preserving signal specificity in high capacity applications.
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 significantly improves signal specificity and allows for high-density analysis of multiple samples without carry-over issues, ensuring accurate results even at low signal intensities.
Implementation Method 1
amplification reaction is performed using a DNA polymerase having 5′->3′-exonuclease activity under conditions that allow removal of (a) a quencher from the labeled oligonucleotide or (b) the distinct second portion from the labeled oligonucleotide
Implementation Method 2
hybridize the pooled labeled oligonucleotides to a solid phase using the respective second portions
Data Source
AI summary
Contemplated systems and methods allow analysis of multiple and distinct patient samples using a labeling scheme that entirely avoids carry-over of a label or reagent to the analytic platform, typically an addressable solid phase. In preferred aspects, a hybridization portion, a fluorophore, and/or a quencher are removed by a 5′-3′-exonuclease activity of a polymerase from a reporter oligonucleotide to so remove the oligonucleotide from the pool of molecules that bind to the solid phase and/or or to provide signal differentiation by removal of a fluorophore or quencher.