Digital PCR Microsatellite Mutation Detection for Low-DNA MSI Analysis

The patent applies segmentation by dividing the DNA sample into numerous individual droplets (thousands to millions of partitions), where each droplet contains at most one target DNA molecule. This digital partitioning enables detection of rare mutant alleles among wild-type alleles by counting positive droplets, achieving sensitivity down to 0.1% mutant allele frequency, far exceeding traditional PCR and NGS methods that require 20% tumor cellularity.

The patent uses fluorescently labeled hydrolysis probes as intermediaries to detect microsatellite instability. These probes specifically bind to microsatellite regions and emit fluorescence signals that indicate the presence of mutant alleles. The probe-based detection system serves as an intermediary mechanism that translates molecular changes into measurable optical signals, enabling highly sensitive detection without requiring complex sequencing equipment.

The patent performs preliminary amplification of target DNA regions before digital partitioning and detection. This pre-amplification step ensures sufficient target material is available for distribution into droplets, enabling sensitive detection of low-abundance mutant alleles. The preliminary action of amplification followed by digital partitioning allows the method to achieve 0.1% sensitivity while maintaining relatively fast analysis time compared to traditional sequential methods.

The patent replaces the mechanical separation and size analysis systems (capillary electrophoresis) with a digital counting system based on fluorescent detection. Instead of physically separating and measuring DNA fragment sizes through electrophoresis, the system uses fluorescent probes and digital droplet counting to detect microsatellite instability, significantly reducing analysis time while improving sensitivity to 0.1%.

The patent applies local quality by designing hydrolysis probes with specific properties tailored for microsatellite detection. The probes are engineered with fluorescent labels and quenchers positioned at specific locations to optimize signal generation upon hybridization to microsatellite regions. This localized optimization of probe structure and fluorescence properties enables reliable detection of mutant alleles at 0.1% frequency while managing the complexity through targeted design rather than system-wide complexity.

The patent changes key detection parameters by using digital droplet partitioning combined with fluorescent signal thresholding. Instead of relying on continuous signal intensity measurements, the system counts discrete positive droplets, changing the detection parameter from analog to digital. This parameter change enables reliable detection at 0.1% sensitivity by statistical counting, overcoming the reliability limitations of traditional continuous measurement methods even with increased probe system complexity.