Multiplex PCR Primer Design for Accurate Gene Abundance Measurement
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Solution Overview
Problem
Conventional PCR techniques face challenges in accurately measuring gene abundance due to differences in primer amplification efficiencies and the plateau phase issue, which complicates the optimization process and increases costs, labor, and time, especially in multiplex PCR where numerous primers are required.
Innovation Solution
A method involving a first primer set that introduces a single additional base sequence to amplification products, allowing for the use of a second primer to uniformly amplify multiple genes, enabling accurate abundance measurement by detecting signals corresponding to these products.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional PCR techniques are used to measure gene abundance, then amplification of target genes can be achieved, but differences in primer amplification efficiencies and the plateau phase issue reduce measurement accuracy
Solution Approach 1:
The patent introduces an intermediary substance (the additional base sequence) that mediates between the different primer sets and the detection system. This intermediary allows uniform amplification of multiple genes by different primers, eliminating the reliability issue of varying primer efficiencies while maintaining the ability to measure gene abundance accurately.
Solution Approach 2:
The patent changes the parameter of primer design by adding a specific additional base sequence to the 5' end of primers. This parameter change enables the primers to uniformly amplify target genes without being affected by the plateau phase, thereby improving both measurement precision and amplification reliability.
2Adaptability or versatility
If multiplex PCR is performed with numerous primers to measure multiple genes, then comprehensive gene abundance data can be obtained, but the optimization process becomes more complex and increases costs, labor, and time
Solution Approach 1:
The patent makes the primers universal by adding a common additional base sequence that enables a single primer set to amplify multiple different target genes. This multi-functional primer design allows comprehensive gene abundance measurement without requiring separate optimization for each gene pair, significantly reducing complexity while maintaining versatility.
Solution Approach 2:
The patent segments the primer structure into two functional parts: the gene-specific binding region and the common additional base sequence. This segmentation allows the primers to maintain gene-specificity for accurate target recognition while sharing a common structure that simplifies the optimization process across multiple genes.
3Quantity of substance
If conventional PCR is used, then amplification products can be obtained, but the plateau phase causes the amplification reaction to end, preventing continuous increase in amplicon amount
Solution Approach 1:
The patent changes the physical parameter of the primer structure by adding an additional base sequence, which fundamentally alters the amplification dynamics. This parameter change prevents the plateau phase from occurring, allowing the amplification reaction to continue indefinitely and the amplicon amount to increase continuously without reaching equilibrium.
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 method allows for more accurate and simpler measurement of gene abundance compared to conventional techniques, avoiding the plateau phase and reducing the complexity and costs associated with primer optimization in multiplex PCR.
Implementation Method 1
a first primer set, which includes at least two types of first primer, each of which is capable of introducing the single additional base sequence to a resulting amplification product
Implementation Method 2
amplifying, in one reaction solution, nucleic acids encoding the at least two genes
Implementation Method 3
determining the abundances of the at least two genes based on detected signals corresponding to the abundances of the at least two types of amplification product
Implementation Method 4
the signals that are used to determine the abundances of the at least two genes are obtained in terms of absorbance or fluorescence values measured at the same wavelength
Data Source
AI summary
The disclosure relates to a method of measuring gene abundance, including obtaining at least two types of amplification product, each of which contains a single additional base sequence and corresponds to each of at least two genes, by amplifying, in one reaction solution, nucleic acids encoding the at least two genes, whose abundances in nucleic acids contained in a subject sample may be different, using a first primer set, which includes at least two types of first primer, each of which is capable of introducing the single additional base sequence to a resulting amplification product and corresponds to each of the at least two genes, and a second primer for amplifying a nucleic acid containing the single additional base sequence; and determining the abundances of the at least two genes based on detected signals corresponding to the abundances of the at least two types of amplification product.