Isothermal Nucleic Acid Amplification via Competitive Stem-Loop Structure
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Solution Overview
Problem
Current nucleic acid amplification methods, such as PCR, require complex temperature control and multiple enzymes, making them costly, prone to contamination, and less suitable for detecting short-strand nucleic acids like 5S rRNA, which are important for genetic analysis.
Innovation Solution
A method for synthesizing nucleic acids under constant temperature conditions using a competitive stem-loop structure with specific oligonucleotides and a single enzyme, such as DNA polymerase, to facilitate efficient and specific amplification of short-strand nucleic acids without the need for complex temperature control or multiple enzymes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If PCR method is used for nucleic acid amplification, then amplification sensitivity is improved, but device complexity and operation difficulty increase due to required temperature control system
Solution Approach 1:
The invention changes the temperature parameter from variable (PCR) to constant (isothermal), using a single temperature step that maintains the reaction mixture at 37°C throughout the amplification process, thereby eliminating the need for complex temperature cycling equipment
Solution Approach 2:
The reaction system uses endogenous RNA polymerase and reverse transcriptase enzymes that naturally function at 37°C, allowing the reaction to proceed autonomously at constant temperature without external temperature control intervention
2Reliability
If PCR method is used for nucleic acid amplification, then amplification sensitivity is improved, but cost increases due to multiple enzymes required
Solution Approach 1:
The invention uses a multi-functional enzyme system where RNA polymerase performs both transcription and the generated DNA serves as template for reverse transcription, reducing the need for separate enzymatic steps and reagents required in traditional PCR
Solution Approach 2:
The invention extracts and utilizes the natural isothermal functionality of RNA polymerase and reverse transcriptase enzymes, removing the need for thermostable DNA polymerase and other heat-cycling dependent components that increase cost
3Productivity
If PCR method is used for nucleic acid amplification, then amplification capability is improved, but contamination risk increases due to susceptibility to external contamination
Solution Approach 1:
The invention segments the amplification process into distinct isothermal stages using specific primer sets, allowing controlled progression of the reaction and reducing the window of vulnerability to contamination compared to continuous heating cycles
Solution Approach 2:
The method uses short-strand specific oligonucleotide primers that are sequence-specific and short-lived in function, providing built-in containment that prevents amplification of non-target sequences and reduces contamination propagation
4Adaptability or versatility
If conventional methods are used for short-strand nucleic acid detection, then detection capability is limited, but method complexity increases due to requirement for complex temperature control
Solution Approach 1:
The invention changes from temperature-cycling parameters to constant temperature parameters, enabling detection of short-strand nucleic acids like 5S rRNA using simple isothermal conditions that do not require complex thermal management
Solution Approach 2:
The reaction system self-regulates at 37°C using the natural optimal temperature of the employed enzymes, making the detection process autonomous and independent of external temperature control equipment
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 method allows for rapid and specific amplification of short-strand nucleic acids, reducing costs and contamination risks, and enabling the detection of target sequences like 5S rRNA, which was previously challenging with existing techniques.
Implementation Method 1
annealing a first oligonucleotide I to the F1c region of the nucleic acid
Implementation Method 2
performing a synthesis step using an F1 region of the first oligonucleotide I as a synthesis starting point
Implementation Method 3
using the nucleic acid provided in the step 1) as a template to synthesize its own complementary strand
Implementation Method 4
a loop can be formed when the Nc region at the 3' end anneals to the N region
Data Source
Figure 1
Figure 2
Figure 3~4
AI summary
The invention discloses a method and a kit for synthesizing a nucleic acid under constant temperature conditions, the method comprising the steps of: 1) providing a nucleic acid, wherein the nucleic acid has an Nc region at the 5' end thereof that can anneal to an N region on the same strand and at the same time has an Nc region at the 3' end thereof that can anneal to the N region on the same strand, and the Nc regions at the 5' end and the 3' end of the nucleic acid have a competitive relationship in annealing to the N region on the same strand; 2) using the nucleic acid of the step 1) as a template to synthesize its own complementary strand with the 3' end of the Nc region which has annealed to the N region as a synthetic starting point; and 3) carrying out complementary strand synthesis by using a polymerase to catalyze strand displacement type complementary strand synthesis reaction, so as to displace the complementary strand synthesized in the step 2). The main advantage of the present invention lies in that rapid amplification of a gene can be achieved using a single enzyme thermostatic system for a short-chain nucleic acid fragment (an ideal fragment can be only 60 bp, which is shorter than the minimum ideal fragment 120 bp of LAMP).