Multiplex PCR Primer Library Design for Non-Target Amplicon Reduction
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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 increase the complexity of Non-Invasive Prenatal Genetic Diagnosis (NPD), particularly in detecting chromosomal abnormalities.
Innovation Solution
A method involving a library of non-immobilized primers that simultaneously hybridize to multiple target loci, with optimized annealing temperatures and extended annealing times to reduce dimer formation, allowing for the simultaneous amplification of multiple nucleic acid regions with high specificity and accuracy.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If multiple oligonucleotide primers are combined in the same PCR reaction to amplify multiple target nucleic acids simultaneously, then assay throughput increases and sample usage efficiency improves, 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 minimize non-target amplification products before conducting the multiplex PCR reaction. This involves evaluating primer combinations in advance to identify sets that are least likely to form primer dimers or amplify non-target sequences, thereby preventing the harmful effect before it occurs during the actual amplification process.
Solution Approach 2:
The patent employs parameter changes by optimizing PCR reaction conditions such as annealing temperature, primer concentrations, and magnesium ion levels to suppress non-target amplification. By adjusting these parameters, the method enables simultaneous amplification of multiple targets while minimizing the formation of harmful byproducts like primer dimers.
2Adaptability or versatility
If the number of primers in multiplex PCR is increased to amplify more targets, then more nucleic acid regions can be analyzed simultaneously, 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 minimize non-target amplification products before conducting the multiplex PCR reaction. This involves evaluating primer combinations in advance to identify sets that are least likely to form primer dimers or amplify non-target sequences, thereby preventing the harmful effect before it occurs during the actual amplification process.
Solution Approach 2:
The patent employs parameter changes by optimizing PCR reaction conditions such as annealing temperature, primer concentrations, and magnesium ion levels to suppress non-target amplification. By adjusting these parameters, the method enables simultaneous amplification of multiple targets while minimizing the formation of harmful byproducts like primer dimers.
3Reliability
If conventional multiplex PCR methods are used for NPD, then chromosomal abnormalities can be detected, but the complexity of the diagnosis process increases due to non-target products
Solution Approach 1:
The patent applies preliminary action by pre-screening and selecting primer pairs based on their ability to minimize non-target amplification products before conducting the multiplex PCR reaction. This involves evaluating primer combinations in advance to identify sets that are least likely to form primer dimers or amplify non-target sequences, thereby preventing the harmful effect before it occurs during the actual amplification process.
Solution Approach 2:
The patent employs parameter changes by optimizing PCR reaction conditions such as annealing temperature, primer concentrations, and magnesium ion levels to suppress non-target amplification. By adjusting these parameters, the method enables simultaneous amplification of multiple targets while minimizing the formation of harmful byproducts like primer dimers.
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, enhancing the sensitivity and specificity of nucleic acid analysis, particularly in NPD, by ensuring that at least 95% of amplified products are target amplicons, thereby improving the accuracy of chromosomal abnormality detection.
Implementation Method 1
a library of non-immobilized primers that simultaneously hybridize to multiple target loci
Implementation Method 2
subjecting the reaction mixture to polymerase chain reaction (PCR) conditions
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.