Multiplex Ligation Probe Microarray for Low-Cost Quantitative Detection
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Conventional real-time fluorescent quantitative PCR methods are limited by high probe synthesis costs, require enzymes with 5'→3' exonuclease activity, and can only achieve 4-5 multiplex detections due to the need for distinguishable fluorophores, complicating optical detection equipment and increasing costs.
Innovation Solution
A method using one pair of primers and multiple pairs of probes for multiplex ligation-dependent amplification, employing a solid phase carrier with surface probes and quenching products to achieve quantitative detection, reducing the need for multiple fluorophores and simplifying optical design.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If multiple separate assays are performed to detect different genetic variations, then detection coverage is improved, but processing time and cost increase
Solution Approach 1:
The patent combines multiple separate genetic variation detection assays into a single multiplexed microarray assay. Different probe sets targeting various genetic variations (SNPs, insertions, deletions) are integrated on one microarray chip, allowing simultaneous detection of multiple genetic markers in a single hybridization experiment, thereby reducing processing time while maintaining comprehensive detection coverage
Solution Approach 2:
The microarray platform is designed with universal applicability to detect multiple types of genetic variations using a single system. The assay can simultaneously analyze different gene regions and variation types through standardized probe design and hybridization protocols, eliminating the need for multiple specialized assays and reducing overall processing time
2Measurement precision
If multiple separate assays are performed to detect different genetic variations, then detection coverage is improved, but cost increases
Solution Approach 1:
The patent consolidates multiple genetic variation detection assays into a single multiplexed microarray experiment. By combining detection of multiple SNPs, insertions, and deletions in one hybridization reaction, the method reduces reagent consumption, labor costs, and overall assay expenses while achieving comprehensive genetic variation detection coverage
Solution Approach 2:
The microarray platform provides universal detection capability for various genetic variations using a single standardized protocol. This multi-functional approach eliminates the need for multiple specialized assays, reducing cumulative costs of reagents, equipment usage, and technical personnel time while maintaining broad detection coverage
3Ease of operation
If conventional hybridization methods are used, then procedure simplicity is maintained, but detection precision and signal-to-noise ratio are insufficient
Solution Approach 1:
The patent segments the detection process into distinct phases: sample preparation, hybridization with specific probe sets, ligation of adapter sequences, and microarray detection. This segmentation allows optimization of each step independently, improving signal-to-noise ratio through targeted probe design and specific ligation conditions while maintaining overall procedural simplicity through standardized protocols
Solution Approach 2:
The patent introduces adapter ligation as an intermediary step between hybridization and detection. The adapter sequence serves as a mediator that enhances specific signal detection by providing a unique molecular tag for amplified detection, thereby improving signal-to-noise ratio without significantly complicating the overall procedure
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 cost-effective multiplex quantitative detection of specific sequences with reduced manufacturing and operational costs, allowing for simultaneous detection of multiple targets without complex optical equipment, and achieving real-time quantification of nucleic acids.
Implementation Method 1
The adapter sequence is biotinylated at the 5' end to facilitate isolation of the hybridized sample using streptavidin magnetic beads
Implementation Method 2
The hybridized sample is denatured and then treated with T7 endonuclease I, which recognizes mismatched base pairs
Implementation Method 3
The extended primer is then used as a template for synthesis of a second strand using a DNA polymerase
Implementation Method 4
Each probe set comprises a probe specific for a wild-type allele and a probe specific for a variant allele
Data Source
Figure 1~2
Figure 3~4
Figure 5(A)~6
AI summary
The present invention provides a multiplex ligation-dependent probe microarray detection. Specifically, a surface probe-based quantitative PCR detection system in the present invention comprises: (a) a solid phase carrier; (b) a first probe; (c) a second probe; (d) a ligase for ligating the first probe and the second probe to form a third probe; (e) a quenching primer pair for amplifying the third probe; and (f) a quenching product capture nucleic acid immobilized on the solid phase carrier and matching a quenching amplification product. The present invention further provides a corresponding detection method and detection apparatus, and applications thereof. The present invention can rapidly, easily, efficiently, and simultaneously perform quantitative detection on multiple target sequences.