Nucleic Acid Quantification via Bridge Oligo Complexes
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Solution Overview
Problem
Current methods for detecting and quantifying genetic variations in samples, such as liquid biopsies, face challenges with specificity, sensitivity, accuracy, throughput, cost, and scalability, particularly in handling complex and impure samples.
Innovation Solution
A method utilizing target-specific nucleic acid probes and a bridge oligo complex for high-throughput detection, involving hybridization, ligation, and high-throughput sequencing to accurately quantify genetic targets in multiple samples, including large volumes and unpurified samples, using a combination of probes that form ligated ligation complexes and unique barcode sequences for identification.
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 quantification experiments required for each genetic target
Solution Approach 1:
The patent combines multiple quantification experiments into a single multiplexed reaction by pooling cDNA from multiple samples and targets, allowing simultaneous quantification of numerous genetic targets across multiple samples in one go, thereby dramatically improving throughput while maintaining accuracy
Solution Approach 2:
The invention uses universal qPCR primers that can amplify multiple different target sequences, allowing a single reaction setup to quantify numerous different genetic targets across multiple samples, eliminating the need for separate experiments for each target
2Productivity
If array qPCR is used to profile multiple genes, then productivity is improved, but device complexity and cost increase due to specialized chips and read-out infrastructure
Solution Approach 1:
The patent uses standard, commercially available qPCR reagents and equipment instead of specialized array chips, effectively copying the successful multiplexing approach to work with conventional infrastructure, thereby reducing device complexity and capital costs while maintaining multi-target profiling capacity
Solution Approach 2:
The invention employs universal primers and a standardized qPCR protocol that can quantify multiple different genetic targets using the same reaction conditions and equipment, making the system universally applicable without requiring target-specific customization of hardware or chips
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 merges quantification of multiple samples and targets into a single pooled reaction, allowing absolute quantification results to be obtained for thousands of samples simultaneously through one experiment, thereby scaling productivity dramatically while maintaining the accuracy of absolute quantification
Solution Approach 2:
The invention performs preliminary pooling and normalization of cDNA samples before the quantification reaction, preparing all samples in advance according to their sequencing depth, which enables the subsequent qPCR reaction to process everything in unison without requiring individual sample processing
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 accurate and massively parallel quantification of nucleic acid targets with high specificity, sensitivity, and scalability, reducing costs and turnaround time, while effectively handling complex and impure samples without prior purification.
Implementation Method 1
allowing the first target specific portion and the second target specific portion of the respective first probe and the second probe to hybridize to essentially adjacent sections on the target sequence, thereby forming a hybridization complex
Implementation Method 2
bringing the hybridization complex in contact with a solid support comprising a second capture moiety and allowing the first capture moiety and the second capture moiety to interact such that the hybridization complexes become linked to the solid support; separating the solid-support-linked hybridization complexes from components of the samples that are not linked to the solid-support
Data Source
AI summary
The present invention disclosure relates to a next generation DNA sequencing method and use for accurate and massively parallel quantification of one or more nucleic acid targets, for example in large volumes of unpurified sample material. More particularly, the invention is related to a method and a kit comprising probes for detecting and quantifying genetic targets in complex samples. The invention includes one or more target-specific nucleic acid probes per genetic target and a bridge oligo or bridge oligo complex.