Snap-back Primers for Nucleic Acid Detection
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Solution Overview
Problem
Current nucleic acid detection technologies face challenges in detecting short target nucleic acids or nucleotides near the 5' or 3' ends, as they require a 'footprint' that is not present in these sequences, limiting their effectiveness.
Innovation Solution
The use of snap-back primers that form 3', 5', or double hairpin structures, which can be used to generate invasive cleavage structures with probes or oligonucleotides, allowing for the detection of target sequences, polymorphisms, and nucleotides, even when the traditional footprint is absent, by employing structure-specific enzymes for cleavage.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional detection methods are used, then detection of target nucleic acid is achieved, but detection of short targets or end-nucleotides is limited due to footprint requirements
Solution Approach 1:
The invention extends detection into a secondary dimension by adding a complementary strand that forms a hairpin structure. This allows the detection system to access target nucleotides that are otherwise inaccessible due to footprint constraints, effectively creating a new dimensional space for probe binding.
Solution Approach 2:
The complementary strand acts as an intermediary element that bridges the gap between the target nucleic acid and the detection probe. By forming a hairpin structure, it creates an accessible interface for probe binding without requiring the target itself to have sufficient length or exposed ends.
2Reliability
If footprint requirements are imposed, then probe stability is improved, but detection of nucleotides near 5' or 3' ends becomes impossible
Solution Approach 1:
The invention creates a copy of the target sequence in complementary form. This copy contains the same informational content but in a format that makes end-nucleotides accessible, allowing probes to bind without requiring the original target to have sufficient footprint.
3Adaptability or versatility
If hairpin structures are formed, then detection of short targets is enabled, but additional structural complexity is introduced
Solution Approach 1:
The invention merges the target sequence information with a complementary strand to create a unified hairpin structure. This consolidation achieves multiple functions simultaneously: preserving target information, creating accessible ends for probing, and maintaining structural stability, without requiring separate complex components.
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 the detection of target sequences and nucleotides, including those near the ends of short RNA sequences, with high specificity and stability at higher temperatures, overcoming the limitations of traditional methods by forming stable hairpin structures that facilitate nucleotide identification.
Implementation Method 1
the 3' end of the oligonucleotide is capable of hybridizing to a complementary sequence within the same oligonucleotide
Implementation Method 2
which may be used to generate invasive cleavage structures with probes or oligonucleotides, allowing for the detection of target sequences, polymorphisms, and nucleotides, even when the traditional footprint is absent, by employing structure-specific enzymes for cleavage
Data Source
AI summary
The present invention provides methods, compositions, and kits comprising snap- back primers used for forming 3' hairpin structures, 5' hairpin structures, and double hairpin structures. The hairpin structures may be used for detecting target sequences (e.g., such as small RNA target sequence), for detecting polymorphisms in target sequences (e.g., such as polymorphisms located near the 5' or 3' ends of the target sequence), or other nucleic acid characterization methods. In certain embodiments, the hairpin structures form invasive cleavage structures (e.g., in combination with a probe or upstream oligonucleotide) which may be cleaved by structure-specific enzymes in order to detect the presence or absence of a particular nucleotide or nucleotide sequence.