Tailed Primer Pairs for Multiplexed Nucleic Acid Detection
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current nucleic acid detection methods face challenges in multiplexed detection, particularly in avoiding cross-hybridization and ensuring specificity, which leads to inconsistent results due to the need for multiple labels and capture antibodies.
Innovation Solution
The use of tailed primer pairs that incorporate a barcode region allows for the multiplexed detection of multiple target nucleic acids through specific hybridization, eliminating the need for multiple labels and capture antibodies by utilizing complementary nucleic acids for recognition.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If multiple labels and capture antibodies are used for multiplexed detection, then the ability to detect multiple target sequences is improved, but cross-hybridization increases and result consistency deteriorates
Solution Approach 1:
The patent introduces a barcode region as an intermediary element that mediates between the target sequence and the capture system. Each target sequence is assigned a unique barcode, and complementary barcodes on capture probes enable specific recognition without cross-reactivity. This intermediary barcode system resolves the contradiction by providing a reliable, consistent mechanism for multiplexed detection that eliminates cross-hybridization issues.
Solution Approach 2:
The detection system is segmented into distinct functional regions: target-specific regions for sequence recognition, barcode regions for identification, and capture regions for immobilization. This segmentation allows each component to perform its function independently and specifically, preventing cross-hybridization while enabling multiplexed detection of multiple targets simultaneously.
2Measurement precision
If different labels are used for each target sequence, then specific recognition is improved, but reagent complexity and cost increase
Solution Approach 1:
The patent employs a universal capture system using complementary barcode sequences that can recognize and capture multiple different target sequences. Instead of requiring unique capture antibodies for each target, a single set of barcode-complementary capture probes can universally capture all targets through their specific barcode regions, reducing reagent complexity while maintaining measurement precision.
Solution Approach 2:
The system uses nucleic acid barcode sequences as information carriers that can be copied and amplified along with the target sequence during PCR. This copying mechanism allows the identification information (barcode) to be replicated without requiring separate labeling reagents for each target, simplifying the reagent system while preserving specific recognition capability.
3Reliability
If capture systems with integrated primers are used, then detection capability is improved, but cross-reactivity increases leading to inaccurate results
Solution Approach 1:
The patent applies local quality by assigning specific functional properties to different regions of the primer: the 5' end contains the barcode region for specific identification, the middle portion contains the target-specific binding region for sequence recognition, and the 3' end contains the capture sequence for immobilization. This localized functional differentiation ensures high specificity at each region, preventing cross-reactivity while maintaining detection accuracy.
Solution Approach 2:
The forward and reverse primers are designed asymmetrically with different barcode sequences and different target-specific regions. This asymmetry ensures that each primer pair is uniquely specific to its target sequence, preventing cross-reactivity between different targets in multiplexed detection while maintaining high detection accuracy for each individual target.
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
This approach enables accurate and consistent multiplexed detection of nucleic acids by minimizing cross-hybridization and improving specificity, thereby enhancing the reliability of nucleic acid detection assays.
Implementation Method 1
a barcode region which comprises a sequence selected from the group consisting of SEQ ID NO: 1 to 8 or a variant thereof capable of specifically hybridizing to a nucleic acid having a sequence which is complementary to SEQ ID NO:1 to 8
Implementation Method 2
the reverse primer contains a target-specific region which is capable of specifically hybridizing to said target nucleic acid and which is labelled in its 5' end with a detectable tag
Implementation Method 3
each type of capture probe contains a region which contains a sequence complementary to a type of barcode region present in the amplicons, said contacting being carried out under conditions adequate for the hybridization between the tail in each type of amplicon and the complementary barcode region in the capture probes
Data Source
Figure 1~3
Figure 4~5
Figure 6~7
AI summary
The present invention relates to a method for the multiplexed detection of nucleic acids in a sample. This is accomplished by using novel sets of primer pairs containing tails which can be selectively captured by complementary tail sequences with negligible cross-hybridization. The invention relates also to reagents, kits and devices used to implement the multiplexed detection method.