Cas9-gRNA Nucleic Acid Isolation via Probe Shielding
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current nucleic acid isolation methods, such as hybrid capture and targeted amplification, suffer from low enrichment efficiency, loss of chemical modifications, and primer cross-reactivity issues, making it difficult to isolate specific target regions without altering the original nucleic acid characteristics.
Innovation Solution
A method utilizing a Type II Cas protein-gRNA complex that specifically binds to target nucleic acid regions, shielding them from external treatments and allowing for high-efficiency isolation with minimal bias, without the need for amplification, and is compatible with downstream sequencing technologies.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If hybrid capture is used to isolate target nucleic acid regions, then enrichment is achieved, but enrichment efficiency is low and at least two rounds of selection are required
Solution Approach 1:
The patent extracts and utilizes the natural complementarity between nucleic acid strands to achieve target region isolation. By designing probes complementary to target sequences, the method directly captures and isolates target nucleic acids without requiring multiple selection rounds or amplification steps, thereby improving enrichment efficiency in a single round.
Solution Approach 2:
The patent introduces probe molecules as intermediaries that bind to target nucleic acid regions through complementarity. These probes serve as mediators to selectively capture and concentrate target sequences, enabling efficient enrichment without the need for repeated selection processes or additional amplification steps.
2Quantity of substance
If targeted amplification is used to increase nucleic acid amount, then quantity is improved, but chemical modifications are lost and bias is generated
Solution Approach 1:
The patent extracts and preserves the original nucleic acid molecules with their chemical modifications intact. By using probe-based capture instead of amplification, the method isolates target regions while maintaining all original molecular characteristics, including chemical modifications, thereby avoiding information loss.
Solution Approach 2:
The patent creates hybridization copies through probe binding rather than enzymatic copying. The probes form complementary hybrid structures with target nucleic acids, allowing isolation without the biased copying process of amplification that introduces errors and loses chemical modifications.
3Quantity of substance
If amplification is used to enrich target regions, then quantity is improved, but primer cross-reactivity limits the number of target regions
Solution Approach 1:
The patent extracts target regions through probe-based capture, allowing multiple target sequences to be isolated simultaneously without interference. Each probe can be designed to bind to specific target regions, enabling multiplexed isolation of numerous targets without the cross-reactivity limitations inherent in primer-based amplification.
Solution Approach 2:
The patent employs a universal probe-based approach that can simultaneously target multiple different nucleic acid sequences. The probes can be designed with specific complementarity to various target regions, allowing a single assay to enrich multiple target regions in parallel, thereby increasing adaptability and versatility.
4Reliability
If whole-genome sequencing is performed to analyze all regions, then completeness is improved, but computing power and storage requirements increase vastly
Solution Approach 1:
The patent extracts and isolates only the relevant target nucleic acid regions from the entire genome before sequencing. By concentrating sequencing efforts on enriched target regions rather than the complete genome, the method maintains analysis completeness for regions of interest while dramatically reducing computing power and storage requirements.
Solution Approach 2:
The patent segments the genome analysis by first isolating specific target regions through probe-based capture. This segmentation allows focused sequencing and analysis of only the necessary portions of the genome, reducing the overall computational and storage burden while maintaining reliability for the analyzed regions.
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 effectively isolates specific nucleic acid target regions while preserving chemical modifications, offering high specificity and efficiency, reducing sample loss, and being compatible with advanced sequencing technologies, thus improving data quality and reducing processing errors.
Implementation Method 1
contacting a population of nucleic acid molecules with at least one Type II Cas protein-gRNA complex, wherein said gRNA comprises a guide segment that is complementary to a sequence comprised in the target region
Data Source
AI summary
The present invention relates to a method for the isolation of a target nucleic acid region. In particular, said method comprises the steps of contacting a population of nucleic acid molecules with at least one Type II Cas protein-gRNA complex, wherein said gRNA comprises a guide segment that is complementary to the sequence comprised in the target region of at least one nucleic acid molecule, thereby forming a Type II Cas protein-gRNA-nucleic acid complex, contacting the population of nucleic acid molecules with at least one enzyme having exonuclease activity, and isolating the target nucleic acid region from the Type II Cas protein-gRNA-nucleic acid complex.