Isothermal Nucleic Acid Detection via Single-Strand Signal Generation
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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, and they are slow, requiring over an hour to perform.
Innovation Solution
A method using restriction enzymes that do not nick, combined with modified dNTPs and additional oligonucleotide probes, allows for rapid amplification without temperature cycling and intrinsic signal detection by producing a detector species through hybridization of two probes, one for detection and one for attachment to a solid material.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Temperature
If SDA or NEAR methods are used for nucleic acid amplification, then isothermal amplification without temperature cycling is achieved, but the amplification time exceeds 1 hour and detection complexity increases due to double-stranded product formation
Solution Approach 1:
The patent modifies the amplification method by changing the enzyme parameters - using a combination of polymerase and nuclease enzymes that work together to create single-stranded products. This parameter change enables faster amplification (reducing time from over 1 hour to approximately 15-30 minutes) while maintaining isothermal conditions, thus resolving the contradiction between temperature control and amplification speed.
Solution Approach 2:
The patent extracts the detection complexity issue by designing the amplification to produce single-stranded products directly, eliminating the need for subsequent strand separation steps. This extraction of the problematic double-stranded intermediate allows for simpler, faster detection while maintaining the isothermal advantage.
2Temperature
If SDA or NEAR methods are used, then isothermal amplification is achieved, but detection requires complex methods and separate process steps due to double-stranded product formation
Solution Approach 1:
The patent removes the complexity of detection by designing the amplification to yield single-stranded products that can be directly detected. This eliminates the need for complex strand separation and detection methods, reducing overall device complexity while maintaining isothermal conditions.
Solution Approach 2:
The patent introduces a nuclease enzyme as an intermediary that works with the polymerase to process the amplified products into single-stranded forms suitable for direct detection. This intermediary enzyme simplifies the detection process by pre-processing the amplification products, eliminating the need for complex additional detection steps.
3Productivity
If restriction enzymes are used in SDA, then strand displacement amplification is achieved, but only a very small number of nicking enzymes are available making application-specific enzyme selection difficult
Solution Approach 1:
The patent achieves universality by using a combination of polymerase and nuclease enzymes that can work together to amplify various target sequences. This multi-functional approach provides greater flexibility in enzyme selection compared to relying on a limited set of nicking enzymes, allowing adaptation to different applications while maintaining high productivity.
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 with efficient signal visualization, suitable for low-cost diagnostic devices, allowing multiplex assays and integration with lateral flow technology for simple, rapid detection.
Implementation Method 1
A strand displacement polymerase extends the 3'-end of each primer 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
hybridisation of the first and second probes to said at least one species within the amplification product produces a detector species
Data Source
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.