Integrated Biochip for Simultaneous Real-Time PCR Quantification and Microarray Qualification
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Solution Overview
Problem
Current molecular diagnostics methods either lack the ability to perform quantitative and qualitative analyses simultaneously or require multiple reaction containers, making them inconvenient and economically inefficient for analyzing multiple gene types.
Innovation Solution
A method that integrates real-time Polymerase Chain Reaction (PCR) and DNA microarray in a single reaction container using a biochip with probes for simultaneous quantitative and qualitative analysis, allowing for the detection of fluorescence signals without opening the container.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If real-time PCR is used for quantitative analysis, then quantitative measurement is possible, but multiple tubes are necessary when examining dozens of gene mutations and genotypes
Solution Approach 1:
The patent combines real-time PCR quantitative analysis with DNA microarray qualitative analysis into a single integrated reaction container. The system uses multiple fluorescent dyes (FAM, HEX, ROX, Cy5) to simultaneously detect multiple gene targets, eliminating the need for separate tubes for different gene examinations while maintaining quantitative measurement precision.
Solution Approach 2:
The reaction container is designed to perform multiple functions: it serves as both a real-time PCR reaction vessel and a DNA microarray substrate. The bottom surface of the container can be coated with capture probes, allowing the same container to be used for both amplification and detection of multiple gene targets, reducing the total number of containers needed.
2Adaptability or versatility
If DNA microarray is used for large-scale qualitative detection, then multiple target probes can be detected at one time, but quantitative analysis is impossible
Solution Approach 1:
The patent merges the qualitative detection capability of DNA microarray with the quantitative measurement capability of real-time PCR. By coating the bottom surface of the reaction container with capture probes and using fluorescently labeled detection probes, the system achieves both multi-target qualitative detection and quantitative measurement within the same container.
Solution Approach 2:
The system uses different fluorescent dyes (FAM, HEX, ROX, Cy5) with distinct emission wavelengths to detect different gene targets. The fluorescent signals are detected in real-time during PCR amplification, enabling both qualitative identification of multiple targets and quantitative measurement of their expression levels or copy numbers.
3Measurement precision
If separate scanner is used to read probe signals, then DNA microarray can be analyzed, but the container must be opened or washed
Solution Approach 1:
The patent integrates the fluorescence detection system directly into the real-time PCR instrument. The same optical detection system used to monitor PCR amplification in real-time is also used to detect fluorescent signals from probes bound to the container bottom surface, eliminating the need for separate scanning equipment and container opening operations.
Solution Approach 2:
The fluorescence signal detection is performed continuously throughout the PCR amplification process. As the PCR reaction progresses and probes hybridize to target sequences, the fluorescent signals are monitored in real-time without interrupting the reaction or requiring container opening, maintaining continuous useful action from amplification to detection.
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 simultaneous quantitative and qualitative analysis of biomaterials in real-time, reducing the need for multiple containers and improving economic feasibility and reliability in molecular diagnosis fields.
Implementation Method 1
a first phosphor for generating a first fluorescence signal
Implementation Method 2
a second phosphor for generating a second fluorescence signal
Implementation Method 3
a first quencher for quenching the first phosphor
Implementation Method 4
a second quencher for quenching the second phosphor
Implementation Method 5
a polymerase having exonuclease activity, wherein the hybridization is only performed between the target genes and the first probe of the complex, and the second probe and the first phosphor are disassembled and released from the complex during elongation by the polymerase having exonuclease activity
Implementation Method 6
hybridization of the target genes, the complex, and the forward and reverse primers in the sample
Data Source
AI summary
A method of quantitatively and qualitatively analyzing a biomaterial in real-time, the method comprising preparing a device for detecting a biomaterial, feeding a complex of first and second probes, a forward primer, a reverse primer, a sample comprising deoxynucleotide triphosphate, a polymerase having exonuclease activity, and a sample comprising target genes, and a reaction solution comprising a buffer into the reaction container, performing polymerase chain reaction comprising denaturation of the target genes in the sample, hybridization of the target genes, the complex, and the forward and reverse primers in the sample, and elongation of the primers through the polymerase having exonuclease activity, allowing for elongation of the second probe on the third probe by the polymerase after hybridizing the released second probe and the third probe fixed to the biochip, detecting a first fluorescence signal by the first phosphor and a second fluorescence signal by the second phosphor.