Fluorescence Cassette Oligonucleotides for Isothermal Nucleic Acid Detection
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Solution Overview
Problem
Current isothermal nucleic acid amplification methods struggle to distinguish between closely related amplification products in multiplex reactions, relying heavily on sequence-specific differences which can limit their ability to differentiate between even closely related targets.
Innovation Solution
The method involves using oligonucleotide primer groups with a fluorescence cassette-specific portion and target-specific portions, along with cassette oligonucleotides labeled with fluorescent and quencher moieties, which hybridize to form a fluorescent quenched pair, allowing for the detection of amplification products through signal generation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If sequence-specific detection methods are used in isothermal amplification, then detection specificity is improved, but the ability to distinguish closely related targets deteriorates
Solution Approach 1:
The primer is segmented into two functional parts: a fluorescence cassette-specific portion and a 3' downstream target-specific portion. This segmentation allows the primer to incorporate a universal fluorescent tag while maintaining target-specific binding capability, enabling differentiation of closely related targets through fluorescence signals.
Solution Approach 2:
A fluorescence cassette oligonucleotide acts as an intermediary that hybridizes to the cassette-specific portion of the primer. This intermediary carries the fluorescent label and enables detection without interfering with the target-specific binding of the primer, thus resolving the contradiction between specificity and versatility.
2Measurement precision
If fluorescent probes are used for detection, then detection sensitivity is improved, but the complexity of the detection system increases
Solution Approach 1:
The fluorescence detection system is merged with the amplification primers themselves. The fluorescent cassette oligonucleotides hybridize to the primer-incorporated cassettes during or after amplification, combining the amplification and detection functions into a unified system that reduces overall complexity.
Solution Approach 2:
The fluorescence cassette-specific portion and the cassette oligonucleotides serve multiple functions: they enable fluorescent labeling, facilitate detection, and maintain compatibility with isothermal amplification. This multi-functionality reduces the need for separate detection components, simplifying the overall system.
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 differentiation of closely related amplification products by generating distinct fluorescence signals, facilitating the detection of multiple targets in isothermal polynucleotide amplification reactions without relying solely on sequence differences.
Implementation Method 1
a first cassette oligonucleotide labelled with a fluorescent moiety (donor moiety) and having a sequence that is capable of hybridisation to the complement of the fluorescence cassette-specific portion of the first oligonucleotide primer of any set in a given oligonucleotide primer group; and a second cassette oligonucleotide labelled with an acceptor moiety (for example a quencher moiety)
Implementation Method 2
the first oligonucleotide primer of each set in the same group contains a fluorescence cassette-specific portion that is capable of hybridising to the complement of the fluorescence cassette-specific portion of the first oligonucleotide primer of any set in the same group
Implementation Method 3
initiating an isothermal polynucleotide amplification reaction in the presence of a strand displacement DNA polymerase thereby generating (if the target polynucleotide is present) a complementary sequence
Data Source
Figure 1A
Figure 1B
Figure 2A~2B
AI summary
A method for the detection of one or more polynucleotide amplification products, the method comprising, in an embodiment, the steps of: a) providing one or more oligonucleotide primer groups, each group comprising: i) a forward primer comprising a fluorescence cassette-specific portion and a 3" downstream target-specific portion; and ii) one or more target-specific primers; b) providing one or more fluorescent quenched pairs, each pair characterised by: i) a first cassette oligonucleotide labelled with a fluorescent moiety and having a sequence that is capable of hybridisation to the complement of the fluorescence cassette-specific portion of the forward primer of a given oligonucleotide primer group; and ii) a second cassette oligonucleotide labelled with a quencher moiety; c) initiating an isothermal polynucleotide amplification reaction in the presence of a strand displacement DNA polymerase thereby generating a complementary sequence to at least a portion of the relevant forward primer, such that the relevant second cassette oligonucleotide is less able to hybridise to the relevant first cassette oligonucleotide, whereby a signal is generated; and d) detecting the signal.