Digital PCR High Titer Quantitation via Internal Standard Competition
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Solution Overview
Problem
Conventional digital PCR methods are impractical for high titer samples with concentrations exceeding 10^6 copies per reaction, as they cannot create a sufficient number of partitions with zero target nucleic acid molecules, making it impossible to apply Poisson statistics for accurate quantitation.
Innovation Solution
The method involves partitioning a sample into subvolumes with a mixture of nucleic acid molecules, amplification reagents, and an internal standard sequence, where some partitions contain zero internal standard molecules, allowing for differential detection of target and internal standard amplicons, and calculating the initial target sequence concentration based on amplification competition.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional digital PCR partitioning is used, then quantitation accuracy is improved through Poisson statistics, but high titer samples cannot be analyzed because insufficient partitions contain zero target molecules
Solution Approach 1:
An internal standard sequence is introduced as an intermediary element that competes with the target sequence for primers during amplification. This mediator allows the system to quantify high titer samples by measuring the competitive inhibition effect, thereby extending the dynamic range while maintaining quantitation accuracy through a different statistical approach than conventional Poisson-based dPCR.
2Adaptability or versatility
If sample concentration is increased to expand dynamic range, then high titer samples can be analyzed, but the number of zero-target partitions decreases making Poisson statistics inapplicable
Solution Approach 1:
The invention changes the fundamental parameter used for quantitation from the presence/absence of target molecules (Poisson statistics) to the degree of competitive inhibition measured by the internal standard's amplification efficiency. This parameter change allows accurate quantitation across a broader concentration range including high titer samples where traditional Poisson methods fail.
3Measurement precision
If more partitions are created to maintain zero-target partitions at high concentrations, then Poisson statistics remain applicable, but partition volume or reaction conditions must be changed
Solution Approach 1:
Instead of creating numerous physical partitions to maintain zero-target conditions, the invention uses a chemical copying approach where the internal standard sequence serves as a proxy for the target sequence. The competitive interaction between target and internal standard copies allows quantitation without requiring extreme partitioning, thereby reducing device complexity while maintaining accuracy.
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 extends the dynamic range of nucleic acid quantitation, enabling accurate measurement of high concentration samples by correlating amplification changes with primer competition, thereby overcoming the limitations of conventional dPCR in high titer samples.
Implementation Method 1
determining the change in amplification of the target sequence in response to primer competition from the internal standard sequence
Implementation Method 2
treating the plurality of partitions under amplification conditions such that the target sequences are amplified to produce detectable target amplicons
Implementation Method 3
The detection reagent comprise a first labeled probe configured to bind to the target sequence, and a second labeled probe configured to bind to the internal standard sequence, wherein the first labeled probe and the second labeled probe are differentially detectable
Data Source
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AI summary
The present invention provides systems, devices, methods, kits, and compositions for nucleic acid analysis using digital PCR. In particular, methods are provided to analyze high titer samples that cannot be divided into a sufficient number of partitions containing zero nucleic acid molecules per partition to allow for Poisson analysis (digital PCR analysis).