Trigger Primer Composition for Genotyping Specificity
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Solution Overview
Problem
Current methods for genotyping, particularly in genes with various variations, face challenges in achieving accurate and sensitive amplification of specific gene regions, often resulting in false positives or negatives due to non-specific hybridization of primers, leading to complex analysis and reduced specificity and sensitivity.
Innovation Solution
A primer composition using trigger primers and amplification primers is designed, where trigger primers are added at low concentrations to initiate PCR and are exhausted early, allowing amplification primers to produce amplification products with identical sequences except for genotype-analyzable sequences, reducing the number of peaks in genotyping assays and improving specificity and sensitivity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If multiple specific primers are used to amplify all gene regions with various variations, then sensitivity is improved, but false positive products are generated due to non-specific hybridization
Solution Approach 1:
The primer composition is segmented into two distinct groups: trigger primers (first group) that specifically bind to variant sequences, and amplification primers (second group) that bind to conserved regions. This segmentation allows the system to first enrich variant-specific products with high specificity, then amplify them with high sensitivity, avoiding the false positives that would result from using a single set of primers for all variants.
Solution Approach 2:
The trigger primers perform a preliminary enrichment step by specifically hybridizing to and amplifying only the variant-containing gene regions before the amplification primers act. This preliminary action ensures that subsequent amplification occurs only on true variant products, eliminating false positives while maintaining high sensitivity for detecting rare variants.
2Measurement precision
If primers are designed for each specific genotype, then specificity is improved, but the complexity of primer design and synthesis increases
Solution Approach 1:
The amplification primers in the second group are designed with universal binding sites that work across all genotypes by targeting conserved regions of the gene. This universal design reduces primer design complexity compared to creating entirely separate primer sets for each genotype, while the trigger primers provide the necessary genotype-specific specificity through their variant-targeting sequences.
3Productivity
If conventional PCR is used to amplify gene regions with various variations, then amplification is achieved, but false negative problems occur due to failing to amplify specific variations
Solution Approach 1:
The trigger primers are designed with local quality optimized for binding to specific variant sequences, while the amplification primers have local quality optimized for binding to conserved regions. This division of functional quality ensures that variant-specific regions are selectively enriched without compromising overall amplification efficiency, thereby eliminating false negatives while maintaining productivity.
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 enables precise amplification and analysis of gene regions with various variations, reducing the complexity of genotyping assays and enhancing the accuracy of genotyping results by minimizing non-specific amplification products.
Implementation Method 1
The primers are designed by selecting a region subjected to be amplified from a gene and determining nucleotide sequences capable of complementarily binding to 3' end sequences of the selected region
Implementation Method 2
a large amount of genetic samples should be obtained before using the methods. Currently, the most common method for obtaining genetic samples is to use the polymerase chain reaction (PCR) method using DNA polymerase
Data Source
Figure 1(A)~1(C)
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AI summary
The present invention provides a primer composition for amplifying a gene region having various variations in a target gene comprising: a first primer group comprising at least one primer specific to at least one template including a genotype-analyzable sequence of a genetically variant base sequence subjected to be analyzed in the target gene; and a second primer group comprising at least one primer designed by selecting one primer having the greatest number of shared bases from the primers of the first primer group and by using the selected primer as a reference, the primer of the second primer group binding specifically to the template or binding complementarily to the template with allowing mismatch of up to three successive bases or 1 to 2 bases in the vicinity of the 3' end of the template.