Normalized Primer Sets for Multiplex Nucleic Acid Signal Equalization
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Solution Overview
Problem
Current STR analysis and genotyping workflows require accurate quantification of template nucleic acid concentrations, which adds time, cost, and risk of contamination and sample loss, especially when dealing with varying template amounts.
Innovation Solution
The method involves a multiplex nucleic acid amplification reaction with normalized signal strength, where either forward primer, reverse primer, or detection probe oligonucleotides are partially labeled, and the ratio of labeled to unlabeled oligonucleotides is adjusted to equalize signal strength across amplicons, eliminating the need for quantification and dilution steps.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If accurate quantification of template nucleic acid concentrations is performed prior to amplification, then signal detection accuracy is improved, but workflow time, cost, and risk of contamination and sample loss increase
Solution Approach 1:
The patent applies preliminary action by incorporating normalized primer sets with predetermined labeled and unlabeled subsets into the amplification reaction before the actual amplification process. This pre-configured primer design eliminates the need for post-extraction quantification steps, as the normalization is built into the reaction system itself, thereby reducing workflow time while maintaining signal detection accuracy
Solution Approach 2:
The invention implements self-service through the normalized primer sets that automatically adjust signal strength based on their internal labeled/unlabeled subset ratios. The system self-regulates the amplification signal without requiring external quantification intervention, allowing the reaction to normalize itself and eliminating additional processing steps that would consume time and resources
2Measurement precision
If quantification and dilution steps are added to the workflow, then signal detection accuracy is improved, but the risk of contamination and sample loss increases
Solution Approach 1:
The patent applies preliminary action by pre-configuring the primer sets with normalized labeled and unlabeled subsets before the amplification reaction. This advance preparation ensures that the signal will be automatically normalized during amplification, eliminating the need for post-extraction quantification and dilution steps that would increase contamination risk and sample loss
Solution Approach 2:
The invention implements self-service through the normalized primer sets that automatically regulate signal strength through their internal composition. The system self-corrects for signal variability without requiring external manipulation of the sample, thereby maintaining reliability by avoiding additional handling steps that could introduce contamination or cause sample loss
3Illumination intensity
If labeled primer/probe oligonucleotides are used in amplification, then signal detection is enabled, but signal strength varies with template concentration
Solution Approach 1:
The patent applies local quality by creating heterogeneity within the primer set itself - combining labeled and unlabeled subsets in specific ratios. This local differentiation within the primer pool allows the system to maintain consistent signal output across varying template concentrations, as the labeled primers provide detection capability while the unlabeled primers modulate the overall signal strength
Solution Approach 2:
The invention implements parameter changes by varying the ratio of labeled to unlabeled primer subsets to optimize signal strength. By adjusting this compositional parameter, the system can maintain consistent fluorescence signal intensity across different template concentrations, effectively decoupling signal strength from template amount variability
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 increases the robustness of STR analysis and genotyping workflows, reduces costs and turnaround time, and minimizes the risk of contamination and sample loss by allowing a broader range of template nucleic acid concentrations without affecting signal detection accuracy.
Implementation Method 1
detecting the one or more signals of the one or more detectable labels
Implementation Method 2
wherein at least one of the oligonucleotides is hybridizable to at least one of the target nucleic acid sequences
Data Source
AI summary
The invention relates to a multiplex nucleic acid amplification reaction comprising at least two separate amplicons, with normalized signal strength, wherein for at least one amplicon either the forward primer oligonucleotides, or the reverse primers oligonucleotides, or the optional detection probe oligonucleotides, are only partially labeled, and the subset amount of labeled primer/probe oligonucleotides to subset amount of unlabeled primer/probe oligonucleotides is adjusted so that the signal strength in both amplicons is equal or nearly equal. Kits and compositions are also claimed.