Single-Stranded Oligonucleotide Probes for Rapid Chromosome Enumeration
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Solution Overview
Problem
Current probes for detecting and enumerating human chromosome 3 are not specific enough and require long assay times due to non-specific binding and repetitive sequences, making them inefficient for diagnostic and research purposes, particularly in lung cancer diagnostics where PIK3CA amplification is a critical target.
Innovation Solution
Development of 18 unique single-stranded oligonucleotide probes that are highly specific to chromosome 3, eliminating the need for blocking DNA and significantly reducing hybridization time, allowing for precise detection and enumeration of chromosome 3 and the PIK3CA gene.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If double-stranded probes with repetitive sequences are used for chromosome 3 detection, then probe availability is improved, but non-specific binding increases and assay time extends to 12-18 hours
Solution Approach 1:
The invention divides the chromosome 3 detection task into multiple independent short probes (typically 3-5 probes of 20-50 nucleotides each) that collectively cover the chromosome region. This segmentation allows each probe to be highly specific and enables parallel hybridization, reducing total assay time from 12-18 hours to 1.5-3 hours while maintaining detection capability
Solution Approach 2:
The invention selects probe sequences from unique, non-repetitive regions of chromosome 3, giving each probe local specificity to avoid binding to centromeric repetitive sequences. This local quality approach eliminates the need for blocking DNA and prevents non-specific binding, thereby reducing assay time without sacrificing probe availability
2Speed
If single-stranded probes are used to reduce assay time, then hybridization speed is improved, but probe specificity deteriorates due to off-target hits
Solution Approach 1:
The invention uses multiple short single-stranded probes (3-5 probes) that collectively cover the target region. Each individual probe is short (20-50 nt) for fast hybridization, but the combination of multiple probes provides robust specificity through cumulative binding strength and reduced off-target effects, achieving both speed and precision
Solution Approach 2:
The invention optimizes probe parameters including length (20-50 nucleotides), concentration, and sequence composition to achieve fast hybridization kinetics while maintaining high specificity. The probes are designed with appropriate Tm values and GC content to ensure specific binding to chromosome 3 sequences without excessive off-target binding
3Measurement precision
If blocking DNA is added to reduce non-specific binding, then measurement precision is improved, but device complexity and procedure steps increase
Solution Approach 1:
The invention extracts and eliminates the need for blocking DNA by selecting probe sequences from unique, non-repetitive regions of chromosome 3. The probes are specifically designed to avoid centromeric repetitive sequences, thereby achieving signal specificity without requiring additional blocking reagents or procedural steps
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
These probes provide discrete, enumerable signals and enable rapid gene copy number enumeration, improving diagnostic efficiency and accuracy in tissue diagnostics, particularly for lung cancer, by achieving specific hybridization in under 3 hours.
Implementation Method 1
Hybridization of chromosome or gene-specific probes has made possible detection of chromosomal abnormalities
Data Source
AI summary
Single-stranded oligonucleotide probes, systems, kits and methods for chromosome enumeration, gene copy enumeration, or tissue diagnostics. The probes are particularly suited for detecting gene amplification, deletion, or rearrangement in tissue samples in a single, dual, or multiplexed assay. The probes exhibit improved performance compared to industry leading dual-stranded probes; particularly in terms of the rate of hybridization and the ability to achieve specific hybridization without blocking DNA.