DNA Quantification via Strand Segmentation and Partitioning
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Solution Overview
Problem
Current DNA quantification methods, such as real-time PCR and digital PCR, face challenges with stochastic sampling errors, especially when dealing with small sample sizes, leading to inaccurate DNA counts due to random fluctuations and background noise, which can impact disease diagnosis and treatment decisions.
Innovation Solution
The method involves dissociating double-stranded DNA into single-stranded DNA and partitioning each strand into separate reactors for amplification and analysis, effectively doubling the number of target molecules, thereby reducing errors associated with small sample sizes and improving the discernibility of DNA signals from noise.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If real-time PCR or digital PCR is used to quantify DNA in small samples, then DNA amounts can be measured, but stochastic sampling errors occur leading to inaccurate counts due to random fluctuations and background noise
Solution Approach 1:
The patent partitions the DNA sample into multiple separate reaction chambers or droplets, where each partition undergoes independent amplification. This segmentation allows for digital counting of DNA molecules by detecting positive partitions, eliminating stochastic sampling errors that occur in bulk amplification methods like real-time PCR. The segmentation principle directly resolves the contradiction by providing both accurate quantification and reliable results even in small samples.
Solution Approach 2:
The patent uses PCR amplification to create copies of target DNA sequences in each partition, generating detectable signals from single-molecule templates. This copying mechanism enables the detection and counting of original DNA molecules through their amplified progeny, achieving precise quantification without requiring large sample sizes while maintaining measurement reliability.
2Quantity of substance
If double-stranded DNA is used directly in partitioning, then the original sample structure is maintained, but the number of target molecules available for analysis is limited
Solution Approach 1:
The patent dynamically converts double-stranded DNA to single-stranded DNA through denaturation before partitioning. This dynamic transformation doubles the number of available target molecules (since each dsDNA yields two ssDNA strands) while maintaining the integrity of the original genetic information. The dynamic state change resolves the contradiction by increasing target molecule quantity without compromising measurement precision.
Solution Approach 2:
The patent changes the physical state parameter of DNA from double-stranded to single-stranded form through controlled denaturation. This parameter change effectively doubles the concentration of target molecules available for partitioning and subsequent detection, thereby improving measurement precision without requiring additional sample material.
3Productivity
If samples with low DNA amounts are analyzed using conventional methods, then analysis can proceed, but the results are affected by random fluctuations and background noise
Solution Approach 1:
By partitioning the sample into numerous independent reactions, the method transforms a continuous analog signal problem into a digital counting problem. Each partition acts as an independent binary outcome (positive/negative), allowing statistical analysis that is inherently more resistant to background noise and random fluctuations, thereby maintaining high measurement precision even with low DNA amounts and high productivity.
Solution Approach 2:
The patent replaces the mechanical/chemical detection of low-abundance DNA signals with a digital counting approach based on partition positivity. This substitution transforms the measurement from an analog intensity-based detection (prone to noise) to a digital presence/absence count (resistant to noise), improving signal discernibility while maintaining analysis throughput.
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 enhances the accuracy of DNA quantification by reducing stochastic sampling errors and allowing for the analysis of samples that would otherwise be rejected, enabling more reliable disease diagnosis and treatment decisions.
Implementation Method 1
Each reactor is then subjected to a PCR reaction, and the presence or absence of the target sequence in the PCR reaction product is detected
Implementation Method 2
qPCR reactions are monitored either using a variety of highly sequence-specific fluorescent probe technologies, or by using non-specific DNA intercalating fluorogenic dyes
Data Source
AI summary
The invention generally relates to methods for increasing the amount of DNA available for analysis when using partitioned samples and parallel processing. For example, double-stranded DNA can be dissociated into two single-stranded components, and the single strands partitioned into different droplets prior to analysis. The disclosed methods are useful for performing digital PCR analysis on samples where the target DNA is not in abundance, for example when the sample originates from a body fluid or an FFPE sample.