Multiplex PCR Primer Library Design for Non-Invasive Prenatal Diagnosis
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current multiplex PCR methods for nucleic acid analysis suffer from the formation of 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 improved sensitivity, specificity, and reduced time and cost are needed.
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 reduce dimer formation and enhance the amplification of target amplicons.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If multiple oligonucleotide primers are combined in multiplex PCR to increase assay throughput, then nucleic acid analysis efficiency is improved, but non-target amplification products such as primer dimers are generated
Solution Approach 1:
The patent applies preliminary action by performing in-silico validation and optimization of primer combinations before actual multiplex PCR. The system uses computational algorithms to predict and eliminate primer-dimer formation, self-complementarity issues, and off-target binding before the physical experiment begins. This pre-screening approach allows multiple primers to be combined in multiplex PCR without generating harmful non-target products, thus resolving the contradiction between increased throughput and harmful amplification products.
Solution Approach 2:
The patent introduces an intermediary computational system that acts as a mediator between primer design and multiplex PCR execution. This in-silico validation platform serves as an intermediary layer that filters out problematic primer combinations using algorithms that predict hybridization patterns, melting temperatures, and dimer formation risks. By inserting this computational intermediary, the system enables safe combination of multiple primers without generating primer dimers or non-target amplification products.
2Adaptability or versatility
If the number of primers is increased to amplify more target loci, then assay coverage is improved, but the risk of generating non-target amplicons increases
Solution Approach 1:
The system performs preliminary computational analysis of all primer combinations to identify and eliminate those that would generate non-target amplicons. Before increasing the number of primers for broader assay coverage, the in-silico validation platform pre-screenes each potential primer against all others in the multiplex, predicting off-target binding and dimer formation. This advance validation enables the system to safely increase primer numbers and assay coverage without proportionally increasing harmful non-target amplicons.
Solution Approach 2:
The patent implements feedback through iterative optimization of primer combinations using computational algorithms. The system analyzes the outcomes of in-silico validation and uses this feedback to refine primer selection, adjusting parameters such as Tm matching, GC content, and sequence composition to minimize non-target amplification. This feedback-driven optimization allows the system to maintain high assay coverage while actively suppressing the generation of non-target amplicons.
3Speed
If standard multiplex PCR conditions are used to amplify multiple targets, then amplification speed is maintained, but sensitivity and specificity are reduced
Solution Approach 1:
The patent applies parameter changes by optimizing specific PCR conditions based on the in-silico validated primer combinations. The system adjusts annealing temperature, extension time, and primer concentrations to match the characteristics of the validated primer set. These parameter optimizations enhance sensitivity and specificity while maintaining amplification speed, resolving the contradiction between fast amplification and precise measurement.
Solution Approach 2:
The system performs preliminary computational determination of optimal PCR parameters before execution. The in-silico platform calculates ideal annealing temperatures, extension times, and primer concentrations based on the specific primer sequences and target characteristics. This pre-determination of optimized parameters allows the physical PCR to proceed quickly with high sensitivity and specificity, eliminating the need for trial-and-error optimization that would slow down the 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 significantly reduces the formation of non-target amplicons, allowing for more accurate and efficient simultaneous amplification of multiple nucleic acid regions, thereby improving the sensitivity and specificity of NPD and reducing the time and cost associated with nucleic acid analysis.
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, to reduce dimer formation and enhance the amplification of 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.