Microsatellite Mutation Detection with Dual-Probe Digital PCR
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Solution Overview
Problem
Current methods for detecting microsatellite instability (MSI) in tumors require high tumor cellularity and are limited in sensitivity, making them inadequate for analyzing low-concentration DNA samples from liquid biopsies.
Innovation Solution
A digital PCR method using two hydrolysis probes, one complementary to the wild-type microsatellite sequence and another to a non-variable region, allows for the detection of MSI by distinguishing fluorescence signals in droplets containing wild-type or mutated microsatellite alleles.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional PCR-based methods are used for MSI detection, then the detection process is simple, but the sensitivity is insufficient and requires a minimum tumor cellularity of 20%
Solution Approach 1:
The patent partitions the PCR solution into numerous discrete droplets, with each droplet serving as an independent reaction chamber. This segmentation enables digital PCR detection, where the presence or absence of fluorescence in individual droplets provides precise quantification of target DNA, achieving detection sensitivity below 0.1% tumor cellularity.
Solution Approach 2:
The patent introduces hydrolysis probes as intermediaries that specifically bind to microsatellite sequences. These probes contain fluorophores and quenchers that generate detectable fluorescence signals only when bound to target DNA, enabling highly sensitive detection of MSI mutations in diluted tumor DNA samples.
2Measurement precision
If next generation sequencing methods are used, then sensitivity improves to 1%, but the complexity and cost increase significantly
Solution Approach 1:
The patent extracts and focuses specifically on the PCR amplification and fluorescence detection steps, eliminating the complex sequencing, data processing, and bioinformatics analysis required by NGS methods. This extraction of the essential detection function achieves comparable or superior sensitivity with much simpler equipment.
Solution Approach 2:
The patent uses disposable droplets as single-use reaction chambers that are generated, used, and discarded in each assay. This approach eliminates the need for expensive, complex sequencing instruments while achieving high sensitivity through simple fluorescence detection in disposable droplet formats.
3Ease of operation
If liquid biopsy samples are analyzed, then non-invasive diagnosis is enabled, but the DNA concentration is very low requiring ultra-high sensitivity detection
Solution Approach 1:
The patent transitions from measuring DNA concentration in a continuous solution to measuring the discrete presence or absence of target DNA in individual droplets. This dimensional change from continuous to discrete measurement enables detection of extremely low DNA concentrations found in liquid biopsy samples through statistical analysis of droplet fluorescence patterns.
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
The method achieves a detection limit 250 times lower than traditional methods, providing high sensitivity and specificity, enabling accurate MSI detection in low-concentration DNA samples, including those from liquid biopsies, and facilitating disease diagnosis, prognosis, and treatment monitoring.
Implementation Method 1
subjecting said DNA sample to a digital polymerase chain reaction (dPCR) in the presence of a PCR solution comprising: a pair of primers suitable for amplifying said target fragment of the DNA sample including said microsatellite sequence
Implementation Method 2
a first oligonucleotide microsatellite (MS) hydrolysis probe, labeled with a first fluorophore, wherein said first MS oligonucleotide probe is complementary to a wild-type sequence including the microsatellite sequence
Implementation Method 3
a first oligonucleotide microsatellite (MS) hydrolysis probe, labeled with a first fluorophore, wherein said first MS oligonucleotide probe is complementary to a wild-type sequence including the microsatellite sequence
Data Source
AI summary
The present disclosure relates to a method for detecting a mutation in a microsatellite sequence locus of a target fragment from a DNA sample, comprising a step of subjecting said DNA sample to a digital polymerase chain reaction (PCR) in the presence of a PCR solution comprising:a pair of primers suitable for amplifying said target fragment of the DNA sample including said microsatellite sequence;a first MS oligonucleotide (MS) hydrolysis probe, labeled with a first fluorophore, wherein said first MS oligonucleotide probe is complementary to a wild-type sequence including the microsatellite sequence;a second oligonucleotide reference (REF) hydrolysis probe, labeled with a second fluorophore, wherein said second oligonucleotide REF probe is complementary to a wild-type sequence of said target DNA fragment which does not include said microsatellite sequence.