Probe-Complex Nucleic Acid Amplification for Accurate Multiplex Quantification
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Solution Overview
Problem
Current methods for detecting and quantifying genetic variations, particularly in samples with weak signals, are cumbersome, laborious, and expensive, lacking specificity, sensitivity, accuracy, throughput, and scalability, especially in complex and large-volume samples.
Innovation Solution
A method using specific oligonucleotide probe complexes with loop and bridge oligonucleotides for amplification, incorporating unique molecular identifiers, enabling high-throughput and accurate quantification through rolling circle amplification and next-generation sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If quantitative PCR is used for genetic target quantification, then measurement precision is improved, but productivity deteriorates due to considerable hands-on time and separate experiments required for each target
Solution Approach 1:
The patent combines multiple target quantification experiments into a single multiplexed assay by pooling DNA samples and performing parallel amplification and sequencing of multiple targets simultaneously, thereby increasing throughput while maintaining quantification accuracy
Solution Approach 2:
The patent creates a universal sequencing library preparation workflow that can quantify multiple genetic targets across many samples using a single experiment setup, making the system multi-functional for both high-throughput screening and accurate quantification
2Productivity
If array qPCR is used to profile multiple targets, then productivity is improved, but device complexity and cost worsen due to expensive chips and read-out infrastructure
Solution Approach 1:
The patent replaces the complex array qPCR hardware system (chips and read-out infrastructure) with a simpler sequencing-based approach using standard library preparation and next-generation sequencing platforms, reducing device complexity and infrastructure cost while maintaining multi-target profiling capacity
Solution Approach 2:
The patent changes the detection parameter from fluorescence-based qPCR readout to sequencing-based detection, allowing multiple targets to be profiled using a single experiment without requiring expensive specialized infrastructure
3Measurement precision
If digital PCR is used for absolute quantification, then measurement precision is improved, but productivity deteriorates due to poor scaling to thousands of samples
Solution Approach 1:
The patent pools DNA samples from multiple sources and combines them into a single sequencing library, allowing absolute quantification of multiple targets across thousands of samples to be performed in parallel through a single sequencing run, thereby achieving both precision and scalability
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
Enables highly sensitive, scalable, and accurate quantification of nucleic acid targets in complex samples, reducing costs and turnaround time while improving specificity and sensitivity.
Implementation Method 1
The probe complexes are allowed to hybridize to target sequences in the sample
Implementation Method 2
enabling high-throughput and accurate quantification through rolling circle amplification
Data Source
AI summary
The present disclosure relates to a method for accurate and massively parallel amplification and quantification of one or more nucleic acid targets, for example in extracted DNA, in large volumes and/or unpurified sample material. The present disclosure includes two target-specific nucleic acid probes per genetic target, a loop oligo and a bridge oligo or bridge oligo complex.