Selective Digital Multiplexing via Blocker Molecules
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Solution Overview
Problem
Current methods for detecting specific nucleic acids, such as circulating tumor DNA, face challenges due to their presence in minor fractions of a sample, alongside abundant non-target DNA and genetic diversity among targets.
Innovation Solution
The use of highly multiplexed digital PCR (dPCR) with blocker molecules that inhibit non-target molecules, allowing for the simultaneous detection of multiple targets in a single readout operation by creating distinct clusters on a 2D plot.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If PCR based assays are used to detect nucleic acids, then amplification of targets is achieved, but detection precision deteriorates when targets are present in small quantities among abundant non-target molecules
Solution Approach 1:
The sample is divided into numerous discrete partitions (droplets or wells), each containing a small volume of the original sample. This segmentation allows rare target molecules to be isolated in individual partitions, separating them from the overwhelming background of non-target DNA. By counting positive partitions rather than measuring bulk signal, the method achieves precise quantification even when targets constitute a tiny fraction of total nucleic acid.
Solution Approach 2:
The method extracts and isolates the target detection function from the bulk sample matrix. Through partitioning, target molecules are physically separated from non-target DNA, and through sequence-specific probes, only target-containing partitions generate signal. This extraction of the detection function from the complex background enables precise measurement of rare targets.
2Productivity
If multiplex detection is implemented to detect multiple targets simultaneously, then productivity increases, but device complexity increases
Solution Approach 1:
The assay uses a universal set of PCR primers that can amplify multiple different target sequences, combined with multiple fluorescently labeled probes specific to different targets. This multi-functional design allows simultaneous detection of numerous targets in a single reaction mixture and single readout operation, achieving high productivity without requiring separate reactions for each target.
Solution Approach 2:
Different targets are detected using probes labeled with different fluorescent dyes that emit at distinct wavelengths. The readout system measures fluorescence intensity across multiple channels, with each channel corresponding to a specific target. This color-based multiplexing allows numerous targets to be distinguished and quantified simultaneously from a single partition set, greatly increasing detection throughput.
3Measurement precision
If blocker molecules are added to suppress wild-type sequences, then measurement precision improves for mutant detection, but device complexity increases
Solution Approach 1:
Blocker molecules serve as intermediary agents that specifically bind to wild-type DNA sequences and prevent them from hybridizing with detection probes or from being amplified. This intermediary action selectively suppresses the abundant wild-type background without affecting mutant targets, thereby enhancing the precision of mutant detection. The blockers are simple oligonucleotides designed to be complementary to wild-type sequences, making them easy to incorporate into the assay.
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 reliable detection and quantification of rare targets, improving resolution and specificity by suppressing wild-type sequences, thus facilitating the identification of multiple variants or mutations in a sample.
Implementation Method 1
Each probe is specific to one of three of the variants and produces fluorescence of a first color or a second color
Implementation Method 2
The blocker can be an oligonucleotide that is perfectly complementary to a nucleic acid that is not the intended target of the dPCR assay. The blocker will bind to the non-target and to amplicon copies thereof.
Data Source
AI summary
The invention provides methods for the detection of molecular targets by digital PCR (dPCR) with a blocker for non-target molecules. Each target is provided with a unique mixture of probes. The blocker binds to a non-target molecule and blocks it from contributing to fluorescence from partitions. Two or more colors of fluorescence intensity are read, and two colors of fluorescence intensity are plotted as a 2D plot. In the plot, different targets contribute well-resolved clusters. Each cluster in the plot essentially lies a long its own radius allowing for radial multiplexing. The use of a blocker selects against the detection of non-target molecules and improves the resolution of radial multiplexing, allowing multiple targets to be detected.