Droplet Digital PCR for cfDNA Integrity and Quantification
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Solution Overview
Problem
Current methods for analyzing circulating cell-free DNA (cfDNA) face challenges in accurately quantifying and assessing DNA integrity, particularly due to pre-analytical factors like fragmentation and the presence of high molecular weight DNA, which can lead to errors in detecting tumor-specific mutations and quantification.
Innovation Solution
A method using droplet digital PCR (ddPCR) with specific primer/probe sets targeting housekeeping genes and employing fluorescent labels to quantify and assess cfDNA integrity, allowing for precise measurement of amplifiable DNA copies and distinguishing between low and high molecular weight fractions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If fluorometric or spectrophotometric methods are used to quantify cfDNA, then quantification can be performed, but accurate assessment of cfDNA integrity and distinction between low and high molecular weight fractions is compromised
Solution Approach 1:
The patent divides the cfDNA population into distinct molecular weight segments (low molecular weight fragments <300bp and high molecular weight DNA) by designing PCR assays with different amplicon sizes. The first assay targets small amplicons that amplify only cfDNA fragments, while the second assay targets large amplicons that amplify both cfDNA and HMW DNA, enabling separate quantification of each segment.
Solution Approach 2:
The patent introduces droplet digital PCR technology as an intermediary method between traditional fluorometric/spectrophotometric quantification and sequencing analysis. This intermediary assay system provides both accurate quantification and integrity assessment by measuring amplifiable copies and calculating integrity metrics that predict sequencing performance.
2Ease of operation
If total cfDNA is measured without distinguishing molecular weight fractions, then quantification is simplified, but downstream sequencing performance is compromised due to HMW DNA interference
Solution Approach 1:
The patent performs preliminary assessment of cfDNA integrity and molecular weight fraction composition before downstream sequencing. By measuring the ratio of amplifiable copies in small versus large amplicon assays, the method predicts sequencing library complexity and identifies samples with excessive HMW DNA that would compromise sequencing performance, allowing for pre-sequencing quality control.
3Productivity
If pre-analytical factors are not controlled, then sample processing is faster, but cfDNA integrity decreases and tumor mutation detection accuracy is reduced
Solution Approach 1:
The patent implements a feedback mechanism where the integrity metric calculated from the two-assay system provides information about the impact of pre-analytical factors on cfDNA quality. This feedback allows identification of samples that have undergone degradation or contamination, enabling quality control decisions about whether to proceed with sequencing or repeat sample collection.
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 provides accurate and precise quantification and integrity assessment of cfDNA, predicting downstream sequencing performance and improving the detection of tumor-specific mutations by accounting for pre-analytical variations and minute DNA amounts.
Implementation Method 1
employing fluorescent labels to quantify and assess cfDNA integrity
Implementation Method 2
droplet digital PCR (ddPCR) with specific primer/probe sets targeting housekeeping genes
Data Source
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AI summary
The present invention provides a method of determining integrity and/or quantity of cell free DNA (cfDNA) in a biological sample comprising amplifying target sequences with at least a first primer/probe set and at least a second primer probe/set, amplifying the target sequences of differing lengths, and monitoring for detection of the labels of the oligonucleotide probes, and determining the integrity and/or quantity of the cfDNA based on the level of detection of the label of the oligonucleotide probe from the first primer/probe set compared to the level detection of the label of the oligonucleotide probe from the second primer/probe set. The present invention also provides methods for generating a library with the cfDNA for sequencing and analysis.