Oligonucleotide Detection Kits via Rolling Circle Amplification
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Solution Overview
Problem
There is a need for effective kits and methods to detect nucleotide sequences, particularly for identifying SNPs associated with diseases or drug responses, as existing genotyping assays face challenges in large-scale analysis and specificity.
Innovation Solution
The method involves using a detection probe with an oligonucleotide tag and a target complement to hybridize with a capture oligonucleotide on a support surface, followed by amplification and labeling to detect the target nucleic acid sequence, utilizing techniques like rolling circle amplification and electrochemiluminescence for sensitive and specific detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If high-density oligonucleotide arrays with hundreds of thousands of probes are used for simultaneous interrogation of many nucleotide sequences, then the ability to perform large-scale analysis is improved, but the device complexity and difficulty of operation increase significantly
Solution Approach 1:
The detection system is divided into modular components: capture oligonucleotides immobilized on support surfaces, detection probes with specific oligonucleotide tags, and standardized amplification/reagent systems. This segmentation allows complex analyses to be performed through coordinated simple modules rather than requiring a single complex high-density array system.
Solution Approach 2:
Oligonucleotide tags serve as intermediary elements that bridge the target nucleic acid sequences and the detection system. These standardized tags enable multiple different target sequences to be detected using a common set of capture oligonucleotides and reagents, simplifying the overall system while maintaining large-scale analysis capability.
2Measurement precision
If detection sensitivity is increased to detect femtomolar concentrations of target analytes, then measurement precision is improved, but the complexity of the detection process and reagent requirements increase
Solution Approach 1:
Capture oligonucleotides are pre-immobilized on support surfaces in specific patterns, and detection probes are pre-designed with specific oligonucleotide tags and amplification sequences. This preliminary preparation enables sensitive detection without requiring complex real-time processing during the actual detection step.
Solution Approach 2:
The detection system replaces complex mechanical detection mechanisms with biochemical amplification processes. Rolling circle amplification and other enzymatic reactions amplify the signal from femtomolar concentrations through biochemical means rather than requiring sophisticated instrumental detection systems.
3Measurement precision
If specificity is improved for detecting particular nucleotide sequences among many variations, then measurement precision is improved, but the complexity of probe design and reagent composition increases
Solution Approach 1:
Specificity is achieved through localized sequence complementarity at specific regions of the oligonucleotides. The capture oligonucleotides and detection probes are designed with specific sequence regions that match particular target sequences, while other regions serve standardized functions. This local specialization enables high specificity without requiring complete redesign of the entire reagent system for each target.
Solution Approach 2:
The oligonucleotide tags and amplification sequences serve multiple functions: they enable specific binding to target sequences, provide standardized interfaces for capture and detection, and facilitate amplification. This multi-functionality reduces the number of separate reagents needed while maintaining high specificity for different target sequences.
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 sensitive detection of nucleotide sequences at femtomolar concentrations, reducing analysis time and improving specificity, allowing for accurate identification and quantification of target analytes in samples.
Implementation Method 1
contacting the sample with a detection probe comprising an oligonucleotide tag, a target complement and a detection oligonucleotide under conditions in which the target complement hybridizes to the target nucleic acid sequence
Implementation Method 2
contacting a support surface on which a capture oligonucleotide is immobilized with a mixture containing the reaction product under conditions in which the oligonucleotide tag of the reaction product hybridizes to the capture oligonucleotide
Implementation Method 3
amplifying the amplification template to form an amplicon comprising one or more nucleic acid sequences comprising detection labeling sites
Implementation Method 4
contacting the amplicon with a detection reagent comprising a label and a nucleic acid sequence that is complementary to the detection labeling sites under conditions in which the nucleic acid sequence of the detection reagent hybridizes to the detection labeling sites
Implementation Method 5
detecting the label bound to the detection labeling sites
Data Source
AI summary
Oligonucleotides, methods and kits are provided for detecting, identifying or quantifying one or more target analytes in a sample as well as methods for immobilizing oligonucleotides onto a support surface.