Isothermal Strand Displacement Control Nucleic Acid Co-Amplification
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Solution Overview
Problem
Current methods for amplifying complex nucleic acids, such as whole genomes, transcriptomes, and bisulfitomes, face challenges in determining the quantity and quality of template nucleic acids, especially with small amounts, due to inhibitors and defects, which affects amplification success and requires improved detection methods.
Innovation Solution
A method involving co-amplification of a control nucleic acid with less than 50% sequence identity to the template nucleic acid, using isothermal strand displacement reactions, allows for the quantification of the control nucleic acid to infer the quantity and quality of the complex template nucleic acid, thereby optimizing amplification conditions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If control nucleic acid is added to compete for amplification resources, then the quantity and quality of template nucleic acid can be determined, but the reaction mixture becomes more complex
Solution Approach 1:
A control nucleic acid is introduced as an intermediary substance that competes for amplification resources (polymerase, primers, dNTPs) with the template nucleic acid. By measuring the amplification efficiency of this control substance, indirect information about the template nucleic acid quantity and quality is obtained. The control nucleic acid acts as a mediator that translates complex template characteristics into measurable signals.
Solution Approach 2:
The amplification of control nucleic acid provides feedback information about the reaction conditions and resource availability. By monitoring how much control nucleic acid is amplified, the system indirectly measures the quality and quantity of template nucleic acid present, creating a feedback loop that enables assessment without direct measurement of the template.
2Quantity of substance
If small amounts of complex template nucleic acid are used, then the amplification process becomes more challenging, but the application requirements demand minimal sample input
Solution Approach 1:
The control nucleic acid is added to the reaction mixture before amplification begins, allowing it to compete for resources during the entire amplification process. This preliminary positioning enables the control to reflect the actual amplification conditions that the template nucleic acid experiences, providing early indication of potential amplification failures due to insufficient template amount or quality.
Solution Approach 2:
The control nucleic acid serves as an intermediary that amplifies even when template amounts are minimal, providing a measurable signal that reflects the reaction conditions. This mediator allows the system to assess whether sufficient template was present for reliable amplification, even when the template itself is too scarce for direct measurement.
3Reliability
If inhibitors are present in the sample, then the amplification efficiency decreases, but real-time detection of inhibitor presence is not possible with current methods
Solution Approach 1:
The control nucleic acid amplification provides continuous feedback about the reaction conditions throughout the amplification process. If inhibitors are present, they will affect both template and control amplification, but the control provides a reference signal that reveals the extent of inhibition. This feedback mechanism enables indirect detection of inhibitor presence and impact on amplification efficiency.
Solution Approach 2:
The control nucleic acid acts as an intermediary that is sensitive to inhibitor presence. Since the control competes for the same resources as the template, any inhibition affecting the reaction will manifest in the control amplification efficiency, allowing indirect measurement of inhibitor impact without requiring direct detection of the inhibitors themselves.
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 accurate determination of the starting amount and quality of complex template nucleic acids, ensuring successful amplification by correlating the amplified control nucleic acid quantities with reaction conditions, thus improving the reliability of whole-genome amplification processes.
Implementation Method 1
co-amplification of the template nucleic acid and the control nucleic acid, wherein the co-amplification takes place isothermally, wherein the co-amplification comprises a strand displacement reaction
Data Source
AI summary
What is described is a method for quantitative and qualitative analysis of complex template nucleic acids to be analyzed. The method comprises the co-amplification of a control nucleic acid with the complex template nucleic acid by means of isothermal strand displacement reaction. The method of the invention further comprises the determination of the amount of amplified control nucleic acid as measure for the determination of the quantity and/or quality of the complex template nucleic acid used. The present invention also relates to a kit for carrying out a method of the invention. Furthermore, the use of the method of the invention or the kit of the invention for standardization of whole-genome, whole-transcriptome and whole-bisulfitome analyses is described.