Multiplex PCR Primer Library Design for Non-Invasive Prenatal Diagnosis
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Solution Overview
Problem
Current multiplex PCR methods for nucleic acid analysis face challenges in reducing non-target amplification products, such as primer dimers, which limit the use of amplified products for further analysis and are particularly problematic in Non-Invasive Prenatal Genetic Diagnosis (NPD), where accuracy and invasiveness are concerns.
Innovation Solution
The method involves contacting a nucleic acid sample with a library of non-immobilized primers that simultaneously hybridize to multiple target loci, followed by primer extension under optimized conditions, including an annealing temperature greater than the melting temperature and extended annealing times, to minimize dimer formation and enhance specific amplicon production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If multiple primer pairs are combined in the same PCR reaction to amplify multiple targets, then assay throughput and efficiency are improved, but non-target amplification products such as primer dimers are generated
Solution Approach 1:
The patent divides the multiplex PCR reaction into two separate reactions: a first PCR reaction that generates initial amplicons, and a second PCR reaction that amplifies those amplicons using universal primers. This segmentation eliminates primer-dimer formation in the second reaction while maintaining high throughput by processing multiple targets across two sequential steps.
Solution Approach 2:
The first PCR reaction performs preliminary amplification of multiple targets using target-specific primers to generate sufficient amplicon templates. This preliminary action ensures that when the second PCR reaction uses universal primers, there is already abundant target material to amplify, preventing the universal primers from binding non-specifically and forming dimers.
2Adaptability or versatility
If the number of primers is increased to amplify more targets, then multiplexing capability is improved, but the risk of generating non-target amplicons increases
Solution Approach 1:
The patent segments the primer functions: target-specific primers in the first PCR ensure specific binding to multiple targets, while universal primers in the second PCR amplify all generated amplicons. This segmentation allows high multiplexing capability through the first PCR's ability to handle many targets, while the second PCR's universal primers maintain high specificity because they only amplify the already-specific first PCR products.
Solution Approach 2:
The first PCR amplicons serve as an intermediary between the target-specific primers and the universal primers. They carry the target-specificity information forward while providing a uniform template structure that the universal primers can reliably bind to, thus mediating between the need for multiplexing and the need for specificity.
3Loss of time
If standard multiplex PCR is used to amplify multiple targets, then analysis time is reduced, but the amplified products are limited for further analysis
Solution Approach 1:
The two-stage PCR process segments the amplification function: the first PCR generates diverse target-specific amplicons, and the second PCR adds universal sequences or structures to these amplicons. This segmentation enables the final products to be suitable for various downstream applications such as sequencing or microarray hybridization, removing the limitations of standard multiplex PCR while maintaining time efficiency.
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 significantly reduces non-target amplification products, improving the sensitivity and specificity of nucleic acid analysis, particularly in NPD, by allowing for the simultaneous amplification of multiple target loci with high accuracy and reduced risk of invasive procedures.
Implementation Method 1
contacting a nucleic acid sample with a library of non-immobilized primers that simultaneously hybridize to multiple target loci
Implementation Method 2
followed by primer extension under optimized conditions, including an annealing temperature greater than the melting temperature and extended annealing times
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.