Solid Phase Nucleic Acid Capture with Strand Displacing Polymerase
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Solution Overview
Problem
Current nucleic acid detection methods face challenges in multiplexing capabilities, with limited ability to amplify multiple targets simultaneously due to non-specific amplifications and primer-dimer artifacts, and existing automation solutions are not suitable for routine clinical analysis.
Innovation Solution
A method utilizing strand displacing polymerases to attach universal priming sequences to target sequences, allowing for robust multiplex amplification in a simple, compact device, which reduces primer-dimer artifacts and enables efficient detection of multiple nucleic acid targets without the need for extensive optimization or denaturation steps.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If standard multiplex PCR methods are used to amplify multiple targets, then multiple nucleic acid targets can be detected simultaneously, but non-specific amplifications and primer-dimer artifacts occur, reducing specificity and yield
Solution Approach 1:
The patent segments the amplification process into two distinct phases: (1) target capture and replication on solid support using strand-displacing polymerase, and (2) subsequent PCR amplification of captured targets. This segmentation isolates the target-specific primers from the amplification phase, preventing primer-dimer formation while maintaining multiplexing capability.
Solution Approach 2:
The patent introduces a solid support as an intermediary between target capture and amplification. Capture probes hybridize to targets on the solid support, and strand-displacing polymerase replicates targets while anchored to the solid support. This intermediary prevents free primers from interacting and forming dimers while enabling specific target amplification.
2Productivity
If multiple pairs of target-specific adapter primers are used for strand displacement amplification, then multiple targets can be amplified, but primer-dimer artifacts are produced, consuming reagents and lowering amplicon yield
Solution Approach 1:
The patent performs preliminary target capture and replication on solid support before amplification. By anchoring targets to the solid support during the replication phase, the system prevents primer-dimer formation in advance, ensuring that reagents are consumed only for productive amplification of captured targets.
3Stability of the object's composition
If universal primers are used for multiplex amplification, then preferential target amplification is avoided and amplification uniformity is improved, but primer-dimer formation by target-specific primers cannot be completely prevented
Solution Approach 1:
The patent extracts target-specific priming activity from the amplification phase by performing target capture and replication on solid support using strand-displacing polymerase. This separation removes the source of primer-dimer artifacts from the amplification reaction, while universal primers are used only in the subsequent amplification phase where they cannot form dimers with target-specific primers.
4Ease of manufacture
If capture probes with RNA/DNA hybrid sequences are used for solid support isolation, then efficient enzymatic digestion by RNase H is achieved, but the method lacks general utility for different types of target molecules and capture sequences
Solution Approach 1:
The patent uses strand-displacing polymerase with universal priming capability that can replicate various target types (DNA, RNA, ssDNA, dsDNA) without requiring target-specific enzymatic digestion steps. The solid support capture and replication system is universally applicable to different target molecules, eliminating the need for RNase H digestion of RNA/DNA hybrids.
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 method allows for reliable and efficient detection and quantification of multiple nucleic acid targets, including small quantities, in a format amenable to automation, reducing primer-dimer artifacts and improving specificity and yield, making it suitable for routine clinical analysis.
Implementation Method 1
processes using strand displacing polymerases to effect strand separation under mild conditions
Implementation Method 2
contacting the sample with at least one oligonucleotide capture probe complementary to a sequence in the target nucleic acid
Data Source
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AI summary
A method and kit for the capture and purification of specific nucleic acids from a sample with affinity capture probes on a solid support and for the replication of said nucleic acids with a strand displacing polymerase, whereby a second primer complementary to a sequence in each of the target nucleic acids distinct from that bound by capture probes is also bound to the nucleic acid targets, and extension of one of the primers on each target effects the separation of the copied nucleic acid strands from the solid support. Incorporation of universal nucleic acid sequences during their replication enables the simultaneous and highly specific amplification of multiple nucleic acid target sequences with minimal production of artifacts.