Template Oligonucleotide Synthesis for Expanded dPCR Quantification
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Solution Overview
Problem
Current digital PCR (dPCR) systems are limited by a 2-log range of quantification precision, requiring multiple assays and large sample sizes, and struggle to accurately quantify nucleic acids with varying concentrations in a single assay due to pipetting errors and inter-assay variability.
Innovation Solution
A method involving the synthesis of template oligonucleotides from nucleic acids using complementary oligonucleotides, followed by PCR amplification, to enhance precision and dynamic range in dPCR, allowing simultaneous quantification of multiple targets.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If conventional dPCR methods are used with limited partitions, then the equipment is simpler and easier to manufacture, but the dynamic range of quantification is limited to about 2-log range
Solution Approach 1:
The invention segments the quantification process into multiple sequential steps within the same partition: initial dPCR amplification, intermediate product detection, and secondary amplification. This temporal segmentation allows the system to achieve extended dynamic range (at least 3 logs) without increasing the spatial number of partitions, thereby maintaining manufacturing simplicity while improving quantification precision
Solution Approach 2:
The method performs preliminary conversion of target nucleic acids into intermediate products before the main quantification amplification. This preliminary action (converting target to intermediate product in a first reaction) prepares the sample in advance, allowing the subsequent amplification to operate over an extended dynamic range without requiring additional partitions or complex equipment modifications
2Measurement precision
If multiple assays are performed to achieve adequate precision, then the quantification accuracy improves, but the sample size requirement increases and the process becomes more costly
Solution Approach 1:
The invention merges multiple quantification functions into a single assay by combining the detection of different concentration ranges within the same partition through sequential amplification steps. The first amplification detects low-abundance targets, while the second amplification of intermediate products detects high-abundance targets, effectively merging the capabilities of multiple assays into one, thereby reducing sample size requirements while maintaining precision
Solution Approach 2:
The method creates a universal quantification system that can handle a wide range of target concentrations (at least 3-log dynamic range) within a single assay configuration. The same partition and reagent system universally quantifies both low and high abundance targets through the dual-amplification approach, eliminating the need for multiple specialized assays and reducing overall sample consumption
3Measurement precision
If multiple chambers are used to average results, then the precision of quantification improves, but the complexity of the analysis process increases and pipetting errors are introduced
Solution Approach 1:
The invention introduces an intermediate product as a mediator between the target nucleic acid and the final detection signal. This intermediate product serves as a bridge that allows the system to detect both low and high abundance targets through a unified amplification pathway, eliminating the need for multiple separate chambers and complex data averaging procedures while maintaining precision
Solution Approach 2:
The system performs self-service quantification within each partition by automatically adapting the amplification process to the target concentration present. The dual-amplification mechanism inherently handles the full dynamic range without requiring external intervention or complex multi-chamber setups, reducing process complexity while maintaining measurement precision through internal self-regulation
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
Extends the dynamic range of dPCR to at least 3 logs, improving quantification precision to 2% relative uncertainty, reducing the need for multiple assays and sample size, and enhancing accuracy in nucleic acid detection.
Implementation Method 1
elongating, by the nucleic acid polymerase, the first and/or second oligonucleotide hybridized by a bridging sequence
Implementation Method 2
hybridizing the first oligonucleotide to the first target region of the target nucleic acid and hybridizing the second oligonucleotide to the second target region
Data Source
Figure 1~2G
Figure 3A~4
Figure 5A~6C
AI summary
The invention relates to a method for converting a nucleic acid target in a sample into a template oligonucleotide, and optionally for detecting and/or quantifying said nucleic acid target. The invention further relates to sets of oligonucleotides and kits for use in a method for converting a nucleic acid target in a sample into a template oligonucleotide, and optionally for detecting and/or quantifying said nucleic acid target.