Multiplex PCR Primer Library Selection to Reduce Primer Dimers
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Solution Overview
Problem
Existing multiplex PCR methods face challenges in reducing the formation of non-target amplification products, such as primer dimers, which limit the use of amplified products for further analysis, particularly in applications like Non-Invasive Prenatal Genetic Diagnosis (NPD), where improved sensitivity and specificity are needed to detect chromosomal abnormalities.
Innovation Solution
A method involving the use of a library of non-immobilized primers that simultaneously hybridize to multiple target loci, with optimized annealing temperatures and primer selection based on dimer formation likelihood, followed by primer extension reaction conditions to produce target amplicons, and subsequent sequencing or hybridization to detect these products.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If multiple pairs of primers are added to the same PCR reaction to amplify multiple target loci simultaneously, then the throughput and efficiency of nucleic acid analysis is improved, but non-target amplification products such as primer dimers are generated which limit the use of amplified products for further analysis
Solution Approach 1:
The patent applies preliminary action by pre-screening and selecting primer pairs based on their likelihood to form dimers before the actual multiplex PCR reaction. A scoring system is used to evaluate potential dimer formation, and primers with high dimer scores are excluded from the multiplex reaction mixture. This advance selection prevents the formation of non-target products during amplification, resolving the contradiction between maintaining high throughput and avoiding harmful byproducts.
2Productivity
If the number of primers in multiplex PCR is increased to amplify more target loci, then assay throughput is improved, but the risk of generating non-target amplicons increases
Solution Approach 1:
The patent employs parameter changes by implementing a quantitative scoring system that evaluates the likelihood of dimer formation based on primer sequence parameters. The scoring system considers factors such as primer concentration, melting temperature, and sequence complementarity. By changing these parameters and selecting primers with optimal scores, the method enables the inclusion of more primer pairs in multiplex reactions while maintaining amplification specificity and reducing non-target product formation.
3Loss of time
If conventional multiplex PCR methods are used to amplify multiple target loci, then time required for nucleic acid analysis is reduced, but the amplified products are limited for further analysis due to non-target amplicons
Solution Approach 1:
The patent implements feedback by using the dimer scoring results to iteratively optimize primer selection for multiplex PCR reactions. The scoring system provides feedback on which primer combinations are likely to produce non-target products, allowing researchers to adjust their primer selections before performing the actual amplification. This feedback mechanism ensures that time-efficient multiplex PCR can be performed while producing clean amplicon libraries suitable for downstream applications such as sequencing and analysis.
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 amplicons, enabling efficient amplification and detection of multiple target loci, enhancing the accuracy and reducing the time and cost of NPD by improving sensitivity and specificity.
Implementation Method 1
a library of test primers that simultaneously hybridize to at least 25 different target loci
Implementation Method 2
subjecting the reaction mixture to primer extension reaction conditions to produce amplified products
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.