Isothermal Nucleic Acid Detection via Cas9 Inhibition
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Solution Overview
Problem
Current methods for detecting nucleic acid mutations and modifications, such as PCR and surveyor assay, face challenges in discriminating between similar nucleic acid sequences and require complex operations for quantitative evaluation, especially in heterozygous mutations, and often necessitate the presence of wild-type DNA for homozygous mutation detection.
Innovation Solution
A method utilizing Recombinase Polymerase Amplification (RPA) under isothermal conditions, where a molecule specifically binds to a nucleic acid with a specific sequence or modification state, allowing for the detection of target nucleic acids by inhibiting amplification of non-target nucleic acids, using molecules like DNA strand cleavage activity-deficient Cas9 or Cas13a proteins with guide RNAs.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If PCR methods using modified oligonucleotide probes with fluorescent substances and quenchers are used to detect gene mutations, then mutation detection capability is improved, but operational complexity increases and quantitative evaluation becomes difficult
Solution Approach 1:
The invention extracts and utilizes the exonuclease activity of DNA polymerase as a separate functional component to cleave the probe, separating the detection function from complex fluorescent labeling and quencher systems. This simplifies the operational procedure while maintaining mutation detection capability.
Solution Approach 2:
The invention replaces the complex fluorescent signal detection system with a simpler colorimetric detection system using chromogenic substrates and enzyme activity measurement, eliminating the need for specialized fluorescent detection equipment and simplifying operational procedures.
2Ease of operation
If surveyor assay using surveyor nuclease is used to detect genome editing, then detection simplicity is improved, but the requirement for control DNA mixing increases device complexity
Solution Approach 1:
The invention extracts and utilizes the endogenous wild-type allele present in heterozygous cells as the control template, eliminating the need to externally mix control DNA. This simplifies the procedure by using what is already available in the sample itself.
Solution Approach 2:
The invention enables the sample to serve itself by using the endogenous wild-type allele within the same sample to provide the control function, eliminating the need for external control materials and reducing procedural complexity.
3Measurement precision
If Sanger sequencing is used to determine nucleotide sequences of individual cells, then direct detection accuracy is improved, but time consumption and cost increase
Solution Approach 1:
The invention replaces the time-consuming Sanger sequencing process with a rapid colorimetric detection system based on enzyme activity measurement. This substitutes a complex, time-intensive sequencing process with a simpler, faster enzymatic reaction that provides sufficient accuracy for mutation detection.
Solution Approach 2:
The invention performs partial sequencing by detecting only the critical mutation sites through targeted probe design, rather than determining the complete nucleotide sequence. This partial action approach achieves the necessary detection accuracy with significantly reduced time and resource investment.
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
Enables simpler and more precise detection of target nucleic acids and evaluation of nucleic acid-binding molecules by reducing the need for thermal cyclers and using the degree of nucleic acid amplification product reduction as an indicator, suitable for detecting gene mutations and modifications.
Implementation Method 1
A method utilizing Recombinase Polymerase Amplification (RPA) under isothermal conditions
Implementation Method 2
using molecules like DNA strand cleavage activity-deficient Cas9 or Cas13a proteins with guide RNAs
Implementation Method 3
allowing for the detection of target nucleic acids by inhibiting amplification of non-target nucleic acids
Data Source
AI summary
The present invention provides a method for detecting a target nucleic acid that discriminates the target nucleic acid from a non-target nucleic acid having a nucleotide sequence or modification state that differs from a portion of the target nucleic acid, the method comprising conducting a nucleic acid amplification reaction using a region in the non-target nucleic acid that differs from the target nucleic acid as a target region, using a region in the target nucleic acid that differs from the non-target nucleic acid as a corresponding target region, using a nucleic acid test sample as a template, and using a primer that hybridizes with both the target nucleic acid and the non-target nucleic acid, with the nucleic acid amplification reaction conducted in the presence of a molecule capable of binding specifically to the target region in the non-target nucleic acid, under temperature conditions under which the molecule can bind to the non-target nucleic acid, and then detecting the target nucleic acid on the basis of the presence or absence of an amplification product.