Integrated Capture and Amplification of Target Nucleic Acid
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Solution Overview
Problem
Next-generation and third-generation sequencing methods require high-quality nucleic acids to function effectively, and the presence of impurities or non-target nucleic acids can lead to misinformation, false positives, and ambiguous results due to contamination.
Innovation Solution
The method involves capturing target nucleic acids using a chimeric capture probe that binds to both the target nucleic acid and an immobilized probe, followed by PCR amplification without dissociation, allowing for the subsequent sequencing of high-quality nucleic acids, even when present in minor proportions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If next-generation and third-generation sequencing methods are used to sequence millions of target templates in parallel, then sequencing efficiency and throughput are improved, but the requirement for high-quality nucleic acids becomes more stringent and contamination has greater harmful effects
Solution Approach 1:
The patent extracts and removes non-target nucleic acids and impurities from the sample before sequencing through specific capture and removal steps. This extraction process isolates the target nucleic acids from contaminants that would otherwise interfere with the sequencing reaction, thereby maintaining high sequencing throughput while eliminating the harmful effects of contamination.
Solution Approach 2:
The patent performs preliminary purification and capture of target nucleic acids before the sequencing process. By conducting capture probes hybridization and selective removal of non-target materials in advance, the system ensures that only high-quality target nucleic acids enter the sequencing reaction, preventing contamination-related failures while maintaining high throughput.
2Manufacturing precision
If capture probes and immobilized probes are used to capture target nucleic acids, then sequencing quality is improved, but the process complexity increases
Solution Approach 1:
The patent combines multiple functions into integrated probe molecules. Capture probes contain both the target-binding region and the immobilization region in a single molecule, eliminating the need for separate capture and immobilization steps. This merging reduces process complexity while maintaining the ability to selectively capture and purify target nucleic acids for high-quality sequencing.
Solution Approach 2:
The capture probes are designed with multi-functionality, serving both as specific binders for target nucleic acids and as anchors for immobilization on magnetic beads or surfaces. This universal design allows a single probe type to perform multiple functions in the purification workflow, simplifying the overall process while ensuring high nucleic acid quality.
3Loss of time
If PCR amplification is performed without dissociation from the capture probe, then time loss is reduced and productivity is improved, but the risk of contamination during transfer steps is eliminated
Solution Approach 1:
The patent merges the capture and amplification steps into a single continuous process. Target nucleic acids are captured on immobilized probes and then amplified in situ without being released or transferred to separate vessels. This integration eliminates transfer steps that could introduce contamination while reducing the overall time required for sample preparation, thereby improving both productivity and reliability.
Solution Approach 2:
The patent maintains continuous action throughout the capture and amplification process. The target nucleic acids remain bound to the capture probes throughout the entire PCR amplification process, eliminating interruptions and transfer steps. This continuity prevents contamination events while minimizing time loss, ensuring both high productivity and low contamination risk.
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 enhances the quality and reliability of nucleic acid sequencing by capturing and amplifying target nucleic acids while minimizing contamination, enabling the identification of mutations, including drug-resistant variants, even at low concentrations.
Implementation Method 1
contacting a target nucleic acid with a capture probe and an immobilized probe, the capture probe comprising a first segment that binds to the target nucleic acid and a second segment that binds to the immobilized probe
Implementation Method 2
the second segment of the capture probe binds to the target, thereby capturing the target nucleic acid
Implementation Method 3
performing a PCR amplification of the captured target nucleic acid without dissociation from the capture probe bound to the immobilized probe
Data Source
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AI summary
There is described a method of preparing a target nucleic acid, comprising contacting a target nucleic acid with a capture probe and an immobilized probe, the capture probe comprising a first segment that binds to the target nucleic acid and a second segment that binds to the immobilized probe, wherein the target nucleic acid binds to the first segment of the capture probe, and the second segment of the capture probe binds to the immobilized probe, thereby forming a capture hybrid capturing the target nucleic acid; and performing a PCR amplification with real time detection of the captured target nucleic acid without a step of dissociating the capture hybrid before initiating thermocycling in the PCR amplification; wherein the PCR amplification is performed in the same vessel as the contacting step.