Strand Invasion DNA Amplification via Separate Oligonucleotide Binding
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Solution Overview
Problem
Current methods for amplifying target nucleic acid sequences by strand invasion are limited in their ability to efficiently amplify longer sequences and are prone to non-specific amplification due to overlapping binding sites of oligonucleotides and primers.
Innovation Solution
The use of strand invasion oligonucleotides that bind at two separate sites on a target nucleic acid sequence, allowing upstream and downstream primers to bind independently and reducing competition with probes, thereby enhancing specificity and efficiency of amplification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If a single strand invasion oligonucleotide is used to open the target duplex, then the target sequence can be accessed for amplification, but the method is limited in amplifying longer sequences and prone to non-specific amplification due to overlapping binding sites
Solution Approach 1:
The patent divides the single strand invasion oligonucleotide function into two separate oligonucleotides, each binding to a distinct site on the target duplex. This segmentation eliminates overlapping binding sites and enables independent binding, thereby improving specificity while extending the amplifiable sequence length through coordinated action of multiple oligonucleotides.
2Productivity
If oligonucleotide binding sites overlap with primer binding sites, then amplification can proceed, but non-specific amplification occurs reducing accuracy
Solution Approach 1:
The patent extracts the strand invasion function from the primer binding function by using separate oligonucleotides for each purpose. The first oligonucleotide mediates strand invasion while the second oligonucleotide and primers handle amplification, thereby eliminating the harmful overlap between binding sites and preventing non-specific amplification.
3Measurement precision
If probes compete with primers for binding sites, then detection can occur, but binding competition reduces amplification efficiency
Solution Approach 1:
The patent resolves the binding competition by transitioning from one-dimensional competition at overlapping sites to multi-dimensional independent binding at distinct sites. The probes, primers, and oligonucleotides bind to different regions of the target sequence, allowing simultaneous detection and amplification without interference.
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 the amplification of longer target sequences and reduces non-specific amplification by allowing independent binding of primers and probes, improving the accuracy and efficiency of the amplification process.
Implementation Method 1
Invasion of the target nucleic acid sequence is mediated by a single strand invasion oligonucleotide, which opens up a target duplex to allow binding of both upstream and downstream primers
Data Source
AI summary
Methods for amplification of a target nucleic acid sequence comprising strand invasion are provided in which strand invasion occurs both at upstream and downstream regions of the target nucleic acid sequence. Further provided are kits and compositions suitable for use in such methods. The methods may comprise amplifying a target nucleic acid sequence comprising a region of unknown sequence, or determining the sequence of a target nucleic acid comprising a region of unknown sequence.