Rapid Isothermal Nucleic Acid Detection With Modified dNTPs
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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 these probes to the amplification product.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Temperature
If SDA or NEAR is used for nucleic acid amplification, then isothermal amplification is achieved without temperature cycling, but the amplification time exceeds 1 hour and the method produces double-stranded products requiring complex detection
Solution Approach 1:
The patent changes the chemical parameters of the amplification system by incorporating modified dNTPs (with non-canonial bases) and using alternative polymerases (Bst, Taq, or other DNA polymerases) to achieve faster amplification rates while maintaining isothermal conditions. This resolves the time contradiction by accelerating the amplification kinetics without changing the temperature protocol.
Solution Approach 2:
The patent uses composite nucleotide structures (modified dNTPs with non-canonial bases combined with standard dNTPs) to enhance amplification efficiency. The modified dNTPs incorporate alternative bases that increase the rate of strand displacement and polymerase activity, thereby reducing amplification time while maintaining the isothermal process.
2Temperature
If SDA or NEAR is used for amplification, then isothermal conditions are maintained, but complex detection methods and separate process steps are required due to double-stranded product formation
Solution Approach 1:
The patent extracts the detection function from separate complex steps by incorporating detection elements directly into the amplification process. Fluorescently labeled probes or other detection moieties are integrated into the amplification reaction mixture, allowing simultaneous amplification and detection without requiring separate post-amplification processing steps or complex equipment.
Solution Approach 2:
The amplification system performs self-detection through the incorporation of fluorescently labeled probes or detection moieties that are part of the amplification mixture. The system detects its own product without external intervention, eliminating the need for separate detection apparatus and simplifying the overall system.
3Stability of the object's composition
If restriction enzymes are used in SDA, then strand separation is achieved, but only a very small number of nicking enzymes are available making it challenging to find enzymes with desired properties
Solution Approach 1:
Instead of using nicking enzymes that can only cleave one strand (limiting enzyme options), the patent uses restriction enzymes that cleave both strands of DNA. This inversion of the enzyme type provides access to a much larger pool of available enzymes with diverse recognition sequences and cleavage characteristics, significantly improving adaptability while maintaining strand separation capability through the action of the polymerase on the nicked strands.
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, allowing efficient signal detection and multiplex assays without complex secondary steps, with amplification rates enhanced by modified dNTPs and a wide range of restriction enzymes available.
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.