Nucleic Acid Detection Through Probe-Mediated Isothermal Signal Release
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Solution Overview
Problem
Existing nucleic acid amplification methods like SDA and NEAR produce double-stranded products, which complicate signal detection and limit their use in low-cost diagnostic devices due to the need for complex detection methods and separate process steps.
Innovation Solution
A method utilizing two additional oligonucleotide probes that hybridize to the amplification product, one for detection and one for attachment to a solid material, allowing for rapid and efficient signal detection without temperature cycling, using restriction enzymes that are not nicking enzymes and incorporating modified dNTPs to block complementary strand cleavage.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If SDA or NEAR is used to amplify nucleic acids, then amplification can be achieved without temperature cycling, but the double-stranded product requires complex detection methods and separate process steps
Solution Approach 1:
The patent extracts the detection function from the amplification process by introducing a separate detection probe that binds to the amplified product. This probe is designed to be cleaved by a restriction enzyme to release a detectable signal, thereby separating the amplification and detection steps while simplifying the overall process.
Solution Approach 2:
The patent introduces a detection probe as an intermediary molecule that bridges the amplified product and the detection signal. This probe binds to the amplification product and, when cleaved by a restriction enzyme, releases a detectable signal, thereby mediating between the amplification step and the detection step.
2Adaptability or versatility
If nicking enzymes are used in NEAR, then strand displacement can be achieved, but only a very small number of nicking enzymes are available making it challenging to find an enzyme with desired properties
Solution Approach 1:
The patent employs asymmetric primer design where one primer contains a restriction enzyme recognition site and the other does not. This asymmetric design allows the use of standard restriction enzymes (which are abundant and well-characterized) instead of rare nicking enzymes, thereby increasing enzyme selection flexibility while maintaining the strand displacement function.
3Temperature
If SDA is used to amplify nucleic acids, then amplification can be performed isothermally, but the method takes over 1 hour to perform limiting its potential in clinical diagnostics
Solution Approach 1:
The patent optimizes reaction parameters including primer concentration, enzyme amount, and incubation time to reduce the amplification time from over 1 hour to a faster duration. By adjusting these parameters while maintaining isothermal conditions, the patent achieves both temperature stability and improved productivity.
4Measurement precision
If additional oligonucleotide probes are added for detection, then detection sensitivity is improved, but the complexity of the assay increases
Solution Approach 1:
The patent merges the detection probe with the amplification process by designing the probe to be incorporated into the amplification reaction mixture. The probe binds to the amplified product during the same reaction, and its cleavage by a restriction enzyme releases a detectable signal, thereby combining detection functionality into the amplification step without requiring separate detection procedures.
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 rapid, sensitive, and specific nucleic acid detection suitable for low-cost diagnostic devices, with enhanced amplification rates and simplified detection processes, including multiplex assays using nucleic acid lateral flow.
Implementation Method 1
A strand displacement polymerase extends the 3'-end of each nick and displaces the downstream DNA strand
Implementation Method 2
a restriction enzyme is used to nick the restriction sites by virtue of its ability to cleave only the unmodified strand of a hemiphosphorothioate form of its recognition site
Implementation Method 3
the cleavage of the reverse complementary strand being blocked due to the presence of one or more modifications incorporated into said reverse complementary strand by the DNA polymerase using the one or more modified dNTP
Implementation Method 4
a first oligonucleotide probe which is capable of hybridising to a first single stranded detection sequence in at least one species within the amplification product
Data Source
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AI summary
The present invention relates to methods for the detection of nucleic acids of defined sequence and kits and devices for use in said methods. The methods employ restriction enzymes, polymerase and oligonucleotide primers to produce an amplification product in the presence of a target nucleic acid, which is contacted with oligonucleotide probes to produce a detector product.