Multiplex PCR and AFM Detection for Nucleic Acid Quantification
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Solution Overview
Problem
Current high-throughput transcriptomic assays are costly, time-consuming, and not sensitive enough for analyzing minute sample quantities, while traditional qPCR is difficult to multiplex, leading to inefficiencies in identifying and quantifying multiple nucleic acid targets.
Innovation Solution
The method involves target-specific multiplex amplification of up to ten targets using PCR followed by single-molecule detection with Atomic Force Microscopy (AFM), allowing for accurate identification and quantification of nucleic acid targets in a single qPCR reaction.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If high-throughput transcriptomic assays (microarrays or RNA-Seq) are used to identify multiple nucleic acid targets, then the identification capability is improved, but the cost, assay time, and input material requirements increase significantly
Solution Approach 1:
The invention segments the high-throughput assay process into a targeted multiplex qPCR approach that focuses on specific gene panels. Instead of analyzing all transcripts, the method divides the task into targeted amplification of selected targets followed by individual detection, reducing assay time from days to hours while maintaining identification capability for clinically relevant genes.
Solution Approach 2:
The invention merges multiple single-target qPCR reactions into a single multiplex reaction by combining multiple primer pairs in one tube. This allows simultaneous amplification and detection of multiple targets in a single assay, reducing the number of reactions from many separate qPCRs to one multiplex reaction, thereby decreasing time and resource requirements.
2Measurement precision
If traditional qPCR is used for multiple targets, then sensitivity is maintained, but the number of reactions and complexity increase
Solution Approach 1:
Multiple single-target qPCR reactions are merged into a single multiplex reaction by combining primer pairs for multiple targets in one tube. The patent demonstrates successful multiplexing of up to 10 targets simultaneously, maintaining the sensitivity of individual qPCR while reducing the number of separate reactions from 10 to 1, thereby simplifying the workflow and reducing complexity.
Solution Approach 2:
The invention creates a universal multiplex qPCR platform that can detect multiple different targets using a single reaction system. The same basic qPCR protocol and detection methodology are applied universally across multiple targets, allowing the system to perform multiple functions (detecting different genes) through a single standardized assay rather than requiring separate specialized reactions for each target.
3Productivity
If multiplex PCR is performed to amplify multiple targets simultaneously, then the number of reactions is reduced, but amplification uniformity and accuracy deteriorate
Solution Approach 1:
The invention applies local quality control by optimizing primer concentrations and amplicon characteristics for each specific target within the multiplex reaction. The patent describes careful selection of primer pairs with similar amplification efficiencies and optimization of individual primer concentrations to ensure uniform amplification across all targets, addressing the non-uniformity problem at the local level of each target rather than treating all targets equally.
Solution Approach 2:
The invention changes key parameters including primer concentrations, amplicon lengths, and annealing temperatures to optimize multiplex amplification uniformity. The patent demonstrates adjustment of primer concentrations for each target and selection of amplicons with similar sizes and GC contents, thereby modifying reaction parameters to achieve balanced amplification across multiple targets and reduce the depletion effect on low-abundance targets.
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 provides a sensitive and cost-effective means to identify and quantify multiple nucleic acid targets, offering higher sensitivity and reduced assay time compared to bulk fluorescent techniques, with the ability to analyze samples at the single-cell level.
Implementation Method 1
performing a polymerase chain reaction (PCR) using primers which produce a uniquely sized amplicon for each of said targets
Implementation Method 2
identifying individual amplicons using atomic force microscopy (AFM)
Data Source
AI summary
A complex mixture is analyzed for multiple nucleic acid sequences (e.g., DNA or RNA sequences) simultaneously by target specific multiplex amplification followed by single molecule detection of amplicons by Atomic Force Microscopy (AFM). The presence or absence of target nucleic acids can be determined from the presence or absence of specific amplicons for those nucleic acids. In addition, quantification of target nucleic acids in the complex mixture is achieved by determination of the numbers of amplicons.