Co-amplifying DNA and RNA for Transcription Normalization
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Solution Overview
Problem
Current methods for normalizing RNA levels are inefficient and inaccurate due to separate extraction and amplification of DNA and RNA, leading to errors in comparison and requiring laborious, time-consuming parallel reactions, and are not well-suited for distinguishing between viable and dead pathogens, especially in cases of antimicrobial resistance and asymptomatic infections.
Innovation Solution
A method for co-amplifying genomic DNA and RNA from a gene along with non-transcribed DNA in a single total nucleic acid sample, using the ratio of amplicon copy numbers or cycle threshold values to normalize transcriptional levels, allowing for simultaneous analysis of both DNA and RNA in a single reaction.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If separate extraction and amplification of DNA and RNA are performed in parallel reactions, then normalization of RNA levels can be achieved, but the process becomes laborious, time-consuming, and error-prone
Solution Approach 1:
The patent combines separate DNA and RNA extraction and amplification processes into a single simultaneous reaction. Total nucleic acid extraction captures both DNA and RNA together, and a single amplification reaction with appropriately designed primers amplifies both nucleic acid types concurrently, eliminating the need for parallel processing while maintaining normalization accuracy
Solution Approach 2:
The patent employs a universal amplification system that can simultaneously amplify both DNA and RNA targets using the same reaction mixture and instrumentation. The primers are designed to specifically target either DNA or RNA sequences, allowing one reaction system to perform multiple functions that previously required separate specialized protocols
2Ease of manufacture
If separate extraction and amplification protocols are used for DNA and RNA, then each can be optimized independently, but errors in comparison between the two protocols increase
Solution Approach 1:
By performing DNA and RNA amplification in the same reaction tube under identical conditions (temperature cycling, enzyme activity, buffer composition), the patent eliminates systematic differences between protocols. Any variations in amplification efficiency are subject to the same conditions, allowing direct comparison without protocol-induced biases
Solution Approach 2:
The patent uses an internal control strategy where a known quantity of exogenous nucleic acid is added to the sample before amplification. This intermediary reference allows normalization and comparison between DNA and RNA measurements while accounting for any residual protocol variations, thereby improving comparison accuracy
3Measurement precision
If conventional normalization methods are used, then RNA levels can be normalized to DNA, but the methods are not well-suited for distinguishing between viable and dead pathogens
Solution Approach 1:
The patent employs different primer designs with specific local characteristics: one set of primers targets highly conserved genomic regions present in both viable and dead pathogens, while another set targets RNA transcripts that are only present in viable, metabolically active pathogens. This local differentiation in target selection enables simultaneous normalization and viability assessment
Solution Approach 2:
The patent utilizes the parameter of nucleic acid stability and persistence. DNA from dead pathogens persists indefinitely and serves as a stable normalization reference, while RNA degrades rapidly after cell death and thus serves as a viability indicator. By measuring both parameters in the same reaction, the system achieves both normalization and viability discrimination
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 provides more accurate and informative results by normalizing RNA levels to genomic DNA, reducing errors and labor, and enabling differentiation between viable and dead pathogens, as well as assessing drug resistance and sensitivity, thereby improving diagnostic accuracy and efficiency.
Implementation Method 1
Methods of in vitro nucleic acid amplification have wide-spread applications in NAAT testing. Such methods include polymerase chain reaction (PCR)
Implementation Method 2
reverse transcription polymerase chain reaction (RT-PCR)
Data Source
AI summary
The present invention provides methods for quantitative data normalisation, and/or ascertaining levels of transcription in cells, organisms, viruses, and the like. The methods can be used in numerous applications including, but not limited to, determining transcriptional upregulation and downregulation, identifying transcriptional perturbation, determining viability/death, and assessing responses to treatment with agents (e.g. resistance or sensitivity to drugs).