Single Cell Nucleic Acid Copy Number Detection via Fluorescent Probes
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Solution Overview
Problem
Digital PCR is not suitable for detecting discrete copy number variations (CNVs) of a specific nucleic acid within a single cell due to its qualitative nature, which limits its ability to quantify the copy number of a specific nucleic acid per single cell in a population.
Innovation Solution
A method involving PCR amplification and quantification of amplicons in reaction compartments using fluorescent probes with different wavelengths to distinguish between target nucleic acid and contaminating cell-free nucleic acid, allowing for the detection of copy number variations in single cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If digital PCR is used for qualitative detection, then presence or absence of template is detected, but copy number quantification per single cell cannot be achieved
Solution Approach 1:
The invention segments the detection process into two distinct phases: (1) PCR amplification phase where target nucleic acid is exponentially amplified, and (2) quantification phase where amplicons are measured during the exponential amplification phase before plateau. This segmentation allows digital PCR to transition from qualitative to quantitative detection by capturing signal intensity data during the exponential phase, thereby resolving the contradiction between maintaining simple digital PCR methodology and achieving precise copy number quantification.
2Reliability
If fluorescent probes with different wavelengths are used to distinguish target from contaminating nucleic acid, then detection accuracy improves, but system complexity increases
Solution Approach 1:
The invention employs fluorescent probes as intermediary molecules that specifically bind to target nucleic acid sequences. Different probes with distinct fluorescence wavelengths serve as mediators to differentiate between target sequences and contaminating cell-free nucleic acid. This intermediary approach enhances detection reliability by providing specific molecular recognition, while the use of well-established fluorescent probe technology keeps the system complexity manageable through leveraging existing biochemical tools.
Solution Approach 2:
The invention utilizes color changes (fluorescence wavelength differences) as a detection mechanism. By employing fluorescent probes that emit at different wavelengths when bound to their respective targets, the system can simultaneously detect and distinguish multiple nucleic acid targets and contaminating material. This optical differentiation method improves reliability through spectral discrimination while maintaining relatively simple detection instrumentation.
3Measurement precision
If quantification is performed during exponential amplification phase, then copy number per single cell is detected, but analysis timing becomes more critical
Solution Approach 1:
The invention implements real-time feedback monitoring of PCR amplification progress through continuous or periodic measurement of fluorescence signal intensity during the amplification cycles. By monitoring the exponential amplification phase in real-time, the system can identify the appropriate quantification window and capture accurate copy number data before the reaction reaches plateau. This feedback mechanism transforms the critical timing requirement into a controllable process parameter, allowing precise quantification while providing operators with temporal guidance for optimal measurement.
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
Enables the precise detection of copy number variations per single cell, including aneuploidy, gene amplification, and deletions, facilitating diagnoses in various conditions such as cancer and prenatal testing.
Implementation Method 1
quantifying an amplicon obtained by the PCR in each of the plurality of reaction compartments during an exponential amplification phase... the plurality of probes being respectively assigned to regions different from each other on the DNA sample... by detecting fluorescence of all of a plurality of wavelengths from the reaction compartment
Data Source
AI summary
The present invention detects the copy number of a specific nucleic acid per single cell in a cell population. This method comprises, in a reaction compartment containing a DNA sample, which is derived from nucleic acids in a single cell, and a PCR system, amplifying a target contained in the DNA sample by PCR. Then, PCR amplicons for each reaction compartment are quantified during the exponential amplification phase.