Single Cell RNA DNA Analysis via cDNA Intermediary
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Solution Overview
Problem
Current methods for analyzing DNA and RNA, particularly in low-cell or limited-sample scenarios, face challenges such as errors in RNA sequencing due to lack of proofreading activity in reverse transcriptases and the need for simultaneous analysis of both DNA and RNA from a single cell or limited nucleic acids.
Innovation Solution
A method involving the use of specific primer pairs and enzymes for simultaneous amplification of target DNA and RNA regions through reverse transcription and PCR, including semi-nested and nested PCR approaches to achieve accurate analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If reverse transcription is used to analyze RNA, then RNA can be converted to cDNA for analysis, but errors are introduced due to lack of proofreading activity in reverse transcriptases
Solution Approach 1:
The patent introduces an intermediary DNA template that is synthesized from RNA through reverse transcription, but then uses DNA polymerase with proofreading activity to amplify and analyze the sequence. This intermediary DNA molecule allows the benefits of RNA analysis while avoiding the proofreading deficiency of reverse transcriptase by using a different enzyme system for the critical amplification and sequencing steps.
Solution Approach 2:
The patent replaces the mechanical system of reverse transcriptase (which lacks proofreading) with a DNA polymerase-based PCR system that possesses proofreading activity. This substitution changes the enzymatic mechanism from one without error correction to one with inherent proofreading capability, thereby reducing error rates in the final analysis.
2Measurement precision
If separate analysis of DNA and RNA is performed, then each can be analyzed with optimized methods, but the process requires multiple steps and increases complexity
Solution Approach 1:
The patent merges the analysis of DNA and RNA into a single integrated workflow. By converting RNA to cDNA through reverse transcription and then using PCR with primers that can amplify both genomic DNA and cDNA, the method combines what would traditionally be separate analytical processes into one unified protocol, reducing the number of steps and overall complexity.
Solution Approach 2:
The patent creates a universal PCR-based method that can analyze both DNA and RNA targets using the same basic protocol and equipment. The primer design and PCR conditions are optimized to work with both genomic DNA and cDNA templates, making the system multi-functional and eliminating the need for separate optimized methods for each nucleic acid type.
3Quantity of substance
If analysis is performed on a low number of cells, then single-cell or limited-sample analysis is achieved, but the amount of target nucleic acids is very limited
Solution Approach 1:
The patent performs preliminary reverse transcription of RNA to cDNA before the PCR amplification step. This preliminary conversion creates a stable DNA copy of the RNA that can then be efficiently amplified by PCR. By performing this action in advance, the method ensures that even very limited amounts of original RNA are converted into amplifiable DNA templates, enabling analysis of single cells or limited samples.
Solution Approach 2:
The patent changes the physical-chemical parameters of the nucleic acid by converting RNA into cDNA. This parameter change (from RNA to DNA) makes the molecule more stable and suitable for PCR amplification, allowing the subsequent exponential amplification to work effectively even when starting with extremely limited amounts of nucleic acid from single cells or limited samples.
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 cost-effective, minimally invasive simultaneous analysis of RNA and DNA from a single cell or limited samples, enhancing the accuracy of gene expression and mutation analysis.
Implementation Method 1
contacting the sample with a reverse primer from a first primer pair, the reverse primer from the first primer pair being directed to a target RNA region, and a reverse transcriptase to effect reverse transcription of the RNA into cDNA
Implementation Method 2
subsequently contacting the sample with: (i) a forward primer from the first primer pair, the forward primer from the first primer pair being directed to a target cDNA region, (ii) a reverse primer and a forward primer from a second primer pair, the reverse primer and forward primer from the second primer pair being directed to a target DNA region, and (iii) a DNA polymerase to simultaneously amplify the target cDNA region and the target DNA region
Data Source
AI summary
A method of simultaneously analyzing RNA and DNA in a sample, the method comprising the step (a) contacting the sample with a reverse primer from a first primer pair directed to a target RNA region to effect reverse transcription of RNA into cDNA with a reverse transcriptase; (b) subsequently contacting the sample with (i) a forward primer from the first primer pair directed to a second cDNA region, (ii) a forward and a reverse primers from a second primer pair targeted to a DNA region, and (ii) a DNA polymerase to simultaneously amplify the target cDNA and target DNA region; and (c) analyzing the amplified target cDNA region and/or amplified target DNA region. Also encompassed are uses of the method to analyze gene expression and mutations, kits comprising primers, enzymes, buffers.