Isothermal DNA Amplification Using Inosine-Containing Primers
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Solution Overview
Problem
Current nucleic acid amplification techniques require multiple steps and different conditions, leading to high background noise and ambiguity in RNA quantification, especially when starting with small amounts of RNA, and often necessitate thermally stable polymerases and equipment, increasing complexity and cost.
Innovation Solution
A method involving reverse transcription of RNA to cDNA using a reverse transcriptase and subsequent amplification with a strand displacement DNA polymerase and Endonuclease V under isothermal conditions, utilizing inosine-containing primers to facilitate nicking and strand displacement without the need for thermal cycling, allowing for robust and sensitive DNA amplification from RNA templates in a single reaction.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If PCR amplification is used to amplify RNA templates, then amplification can be achieved, but multiple temperature cycling steps are required and thermally stable polymerases are needed, increasing equipment requirements and complexity
Solution Approach 1:
The invention changes the temperature parameter from cyclic variation to constant isothermal condition (40-65°C). By using a strand displacement DNA polymerase that functions optimally at isothermal conditions, the method eliminates the need for thermal cycling equipment while maintaining amplification reliability through the cooperative action of reverse transcriptase, endonuclease, and DNA polymerase at a single temperature
Solution Approach 2:
The invention extracts and removes the thermal cycling step from the amplification process. By using inosine-containing primers that can be nicked by endonuclease at isothermal conditions, the method separates the amplification mechanism from the thermal cycling requirement, eliminating the need for complex temperature control equipment
2Reliability
If multiple step amplification methods are used for RNA templates, then amplification can be achieved, but the process becomes complex with high background noise and ambiguous results
Solution Approach 1:
The invention merges reverse transcription and DNA amplification into a single isothermal reaction step. By using inosine-containing primers that serve dual functions and the cooperative action of reverse transcriptase, endonuclease, and DNA polymerase, the method combines multiple functions into one streamlined process, reducing background noise and eliminating ambiguous intermediate steps
Solution Approach 2:
The inosine-containing primers serve multiple functions: they serve as templates for reverse transcription, contain recognition sites for endonuclease nicking, and enable strand displacement for amplification. This multi-functionality reduces the number of separate reagents and steps required, simplifying the overall process while maintaining reliability
3Quantity of substance
If RNA templates are amplified using conventional methods, then amplification can be achieved, but background noise increases and quantification accuracy decreases
Solution Approach 1:
The invention changes the reaction temperature parameter to isothermal conditions (40-65°C), which optimizes the activity of reverse transcriptase, endonuclease, and DNA polymerase simultaneously. This parameter change reduces non-specific amplification and background noise while maintaining high amplification yield, thereby improving quantification accuracy
Solution Approach 2:
The inosine-containing primers act as intermediaries that facilitate specific recognition and nicking by endonuclease. This intermediary mechanism ensures specific amplification of the target sequence, reducing non-specific background noise and improving the accuracy of RNA quantification while maintaining high yield
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 efficient, sensitive, and reproducible amplification of DNA from RNA templates without denaturation, reducing background noise and simplifying the process, while maintaining high specificity and sensitivity, thus overcoming the limitations of existing methods.
Implementation Method 1
reverse-transcribing a ribonucleic acid (RNA) template to form a cDNA using a first reaction mixture comprising RNA template, at least one primer capable of hybridizing to the RNA template, a reverse transcriptase and deoxynucleoside triphosphates (dNTPs)
Implementation Method 2
DNA polymerases add nucleotides to the 3′ end of a pre-existing DNA strand resulting in 5′→3′ elongation in a template-directed fashion to create a complementary strand
Implementation Method 3
Endonuclease V cleaves the second (or third) phosphodiester bond 3′ to the inosine in the same strand, leaving a nick with a 3′-hydroxyl and 5′-phosphate
Implementation Method 4
at least one inosine-containing primer and a nuclease that is capable of nicking DNA 3′ to an inosine residue of the primer
Data Source
AI summary
A method of amplifying RNA template is provided. The method comprises reverse-transcribing a ribonucleic acid (RNA) template to form a cDNA using a first reaction mixture comprising RNA template, at least one primer capable of hybridizing to the RNA template, a reverse transcriptase and deoxynucleoside triphosphates (dNTPs); and amplifying the cDNA to form an amplified product using a second reaction mixture comprising at least one strand displacement DNA polymerase, at least one inosine-containing primer and a nuclease that is capable of nicking DNA 3′ to an inosine residue of the primer. The method is accomplished under an isothermal condition without denaturing the cDNA template. A method of quantifying RNA template in a sample and a method of detecting RNA template in a sample are also provided.