Allele Detection Probe Competitor Assay
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Solution Overview
Problem
Current amplification assays for detecting allelic forms of a target sequence, such as mutant and wild-type forms, are inefficient and prone to false positives due to the need for multiple probes and the difficulty in distinguishing between rare mutant sequences, especially when they co-occupy the same partition, leading to reduced sensitivity and accuracy.
Innovation Solution
The method involves creating partitions with a probe capable of binding to both allelic forms and a competitor that selectively binds to the wild-type form, blocking probe binding and reducing false positives, allowing for the detection of variant forms using the same probe and increasing sensitivity by distinguishing based on signal strength and partition occupancy.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If a different mutant probe is used to detect each different mutant form, then the specificity for detecting each mutant form is improved, but the device complexity and cost increase
Solution Approach 1:
The patent employs a single universal probe that can detect multiple different mutant forms of a target sequence. This probe is designed with binding specificity that allows it to recognize various mutations without requiring separate probes for each mutant type, thereby reducing device complexity while maintaining detection precision through the use of a competitor oligonucleotide to enhance discriminatory power.
Solution Approach 2:
The patent introduces a competitor oligonucleotide as an intermediary element that binds to the target sequence and modulates probe binding. This competitor acts as a mediator that enhances the probe's ability to distinguish between different mutant forms, allowing a single probe to achieve high specificity across multiple mutant types without requiring multiple specialized probes.
2Measurement precision
If the mutant form is rare in a sample, then the sensitivity for detecting the mutant form should be increased, but false positives from wild-type form detection swamp out true positives
Solution Approach 1:
The patent applies preliminary anti-action by using a competitor oligonucleotide that pre-binds to the wild-type target sequence before the probe can bind. This preliminary binding by the competitor prevents the probe from binding to abundant wild-type sequences, thereby suppressing false positive signals before they can occur and allowing rare mutant forms to be detected with high sensitivity and reliability.
Solution Approach 2:
The patent employs local quality by designing the competitor oligonucleotide with sequence specificity tailored to the wild-type target region. This localized complementary binding creates a specific interaction that selectively blocks probe binding to wild-type sequences while leaving mutant sequences accessible, thereby enhancing sensitivity for rare mutants without increasing false positives from wild-type detection.
3Adaptability or versatility
If partitions contain both mutant and wild-type forms, then the ability to detect co-occurring alleles is improved, but the difficulty in distinguishing between them increases
Solution Approach 1:
The patent uses a competitor oligonucleotide as an intermediary that selectively binds to wild-type sequences in partitions containing both mutant and wild-type forms. This mediator creates a differential binding scenario where the probe is blocked from binding to wild-type sequences but remains free to bind to mutant sequences, thereby enabling clear distinction between co-occurring alleles and simplifying detection despite the presence of multiple allele types.
Solution Approach 2:
The patent employs signal differentiation (analogous to color changes) by using a labeled probe that produces a detectable signal only when bound to mutant sequences. The competitor-induced blocking of wild-type binding creates a signal contrast that allows easy visual or instrumental distinction between partitions containing mutant alleles versus those containing only wild-type alleles, thereby simplifying the detection of co-occurring alleles.
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 enhances the sensitivity and accuracy of detecting rare mutant sequences by reducing false positives and enabling the use of the same probe for multiple allelic forms, improving the detection of variants and wild-type forms in amplification assays.
Implementation Method 1
a same probe capable of binding specifically to each of the first and second allelic forms of the target
Implementation Method 2
a competitor configured to bind selectively to the second allelic form relative to the at least one first allelic form and to block binding of the probe to the second allelic form
Implementation Method 3
detecting a signal from a label of the probe
Data Source
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Figure 5
AI summary
Methods and compositions for detecting an allelic form of a target. In an exemplary method, partitions may be created that collectively contain at least one first allelic form and a second allelic form of a target. Each partition may contain (i) a same probe capable of binding specifically to each of the first and second allelic forms of the target and (ii) a competitor configured to bind selectively to the second allelic form relative to the first allelic form and to block binding of the probe to the second allelic form. The first allelic form of the target may be amplified in the partitions. A signal may be detected from a label of the probe while the label is contained by the partitions. A number of partitions that are positive (or negative) for the at least one first allelic form may be determined based on the signal.