Multiplex PCR Primer Library Selection for Non-Target Amplicon Reduction
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Solution Overview
Problem
Current multiplex PCR methods face challenges in reducing non-target amplification products, such as primer dimers, which limit the use of amplified products for further analysis and assays, especially when amplifying multiple nucleic acid regions simultaneously.
Innovation Solution
The method involves using a library of test primers that simultaneously hybridize to a large number of target loci, with primer extension reactions under PCR conditions, and selecting primers based on undesirability scores to minimize dimer formation, allowing for the simultaneous amplification of thousands of target loci with reduced primer dimers.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If multiple pairs of primers are added to the same PCR reaction to amplify multiple target nucleic acids simultaneously, then assay throughput is increased and experimental procedures are simplified, but non-target amplification products such as primer dimers are generated
Solution Approach 1:
The patent applies preliminary action by pre-screening and selecting primer pairs based on their ability to form dimers or non-target products. Before conducting the multiplex PCR reaction, the method evaluates potential primer combinations and eliminates those likely to generate harmful byproducts, thereby preventing rather than treating the problem of non-target amplification.
Solution Approach 2:
The patent utilizes parameter changes by optimizing PCR reaction conditions such as temperature, primer concentration, and cycle parameters to favor specific amplification over primer dimer formation. By adjusting these parameters, the method maintains high throughput while reducing the generation of non-target products.
2Adaptability or versatility
If the number of primers is increased to amplify more target loci, then multiplexing capability is improved, but the risk of generating non-target amplicons increases
Solution Approach 1:
The patent applies preliminary action by pre-screening and selecting primer pairs based on their ability to form dimers or non-target products. Before conducting the multiplex PCR reaction, the method evaluates potential primer combinations and eliminates those likely to generate harmful byproducts, thereby preventing rather than treating the problem of non-target amplification.
Solution Approach 2:
The patent utilizes feedback by using computational algorithms to predict and evaluate primer interactions before the actual PCR reaction. The method provides feedback on which primer combinations are likely to produce non-target amplicons, allowing researchers to select optimal primer sets that maximize multiplexing capability while minimizing harmful byproducts.
3Reliability
If non-target amplicons are reduced through improved primer selection, then the use of amplified products for further analysis is enhanced, but the complexity of primer selection process increases
Solution Approach 1:
The patent replaces manual primer selection with computational algorithms and automated screening methods. Instead of relying on manual evaluation of primer combinations, the method uses computer-based predictions and automated assays to identify optimal primer sets, thereby reducing the complexity and time required for primer selection while maintaining or improving product quality.
Solution Approach 2:
The patent applies self-service by designing primer sets that are self-evaluating through their sequence characteristics. The method incorporates features such as unique molecular identifiers and designed sequence properties that allow the primers themselves to indicate their suitability for multiplexing, reducing the need for external evaluation and simplifying the selection process.
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 the efficient amplification of a large number of target loci with minimal primer dimers, enhancing the accuracy and throughput of nucleic acid analysis, suitable for applications like non-invasive prenatal genetic diagnosis and forensic analysis.
Implementation Method 1
contacting the nucleic acid sample with a library of test primers that simultaneously hybridize to at least 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40,000; 50,000; 75,000; or 100,000 different target loci
Implementation Method 2
subjecting the reaction mixture to primer extension reaction conditions to produce amplified products that include target amplicons
Data Source
AI summary
The invention provides methods for simultaneously amplifying multiple nucleic acid regions of interest in one reaction volume as well as methods for selecting a library of primers for use in such amplification methods. The invention also provides library of primers with desirable characteristics, such as minimal formation of amplified primer dimers or other non-target amplicons.