Solid Phase Negative Enrichment of Target Nucleic Acids
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Solution Overview
Problem
Current disease detection methods, particularly in molecular genetics, are time-consuming and error-prone due to complex and invasive sample preparation procedures, often requiring large biological samples and expensive kits, which can lead to delayed treatment and improper intervention.
Innovation Solution
The use of a complex of Cas endonuclease and guide RNA bound to a solid surface or particle to selectively capture and enrich target nucleic acid from biological samples, reducing the need for extensive sample preparation and enabling detection of low-abundance DNA sequences, such as circulating tumor DNA.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If complex sample preparation protocols are used to prepare biological samples for sequencing, then the detection accuracy and reliability are improved, but the time required for sample preparation increases significantly and human error increases
Solution Approach 1:
The invention extracts and isolates the essential function of sample preparation by using magnetic beads functionalized with capture probes that specifically bind to target nucleic acids. This extracts the target DNA/RNA directly from the biological sample without requiring complex multi-step preparation protocols, thereby reducing preparation time while maintaining detection accuracy through specific magnetic separation and washing steps.
Solution Approach 2:
The invention introduces magnetic beads functionalized with capture probes as an intermediary between the biological sample and the sequencing analysis. These magnetic beads serve as a mediator that selectively captures target nucleic acids through hybridization, allowing for simplified separation and concentration of targets without requiring complex sample preparation steps, thus reducing time while preserving detection accuracy.
2Reliability
If complex sample preparation protocols with multiple steps are used, then the detection reliability is improved, but the probability of human error increases
Solution Approach 1:
The invention extracts the critical separation and purification functions into a single magnetic bead-based step. By functionalizing magnetic beads with capture probes that specifically bind target nucleic acids, the method consolidates multiple preparation steps into one operation: add magnetic beads to sample, incubate for hybridization, apply magnetic field for separation, and proceed to analysis. This maintains detection reliability through specific binding while dramatically simplifying operation and reducing human error opportunities.
Solution Approach 2:
The magnetic beads perform self-service by automatically separating from the sample matrix through magnetic field application after hybridization. The beads with bound targets self-concentrate and can be directly transferred to sequencing platforms without requiring manual manipulation or complex handling steps, thereby simplifying operation while maintaining reliability through consistent, reproducible separation.
3Quantity of substance
If large biological samples are used to extract sufficient DNA for sequencing, then the DNA quantity for analysis is improved, but the invasiveness and pain to the patient increases
Solution Approach 1:
The invention employs highly efficient capture probes that enable continuous and thorough binding of target nucleic acids from small input samples. The magnetic beads with optimized capture probes continuously capture targets during incubation, maximizing DNA yield from minimal sample input. This allows sufficient DNA quantity for sequencing to be obtained from small, less invasive biological samples rather than requiring large sample volumes that would increase patient invasiveness.
Solution Approach 2:
The invention changes the parameters of the capture system by using magnetic beads with high surface area and optimized probe density, which dramatically increases the capture efficiency per unit volume of sample. This parameter optimization allows sufficient DNA quantity to be extracted from small sample volumes, thereby reducing the need for large, invasive sample collections while maintaining adequate DNA quantity for sequencing analysis.
4Difficulty of detecting and measuring
If next-generation sequencing methodologies are used to detect variants, then the detection capability is improved, but the reproducibility of detected variants decreases
Solution Approach 1:
The invention performs preliminary enrichment and purification of target nucleic acids using magnetic beads with specific capture probes before sequencing. By pre-concentrating and purifying the targets of interest, the method reduces background noise and off-target sequences that can cause false variants in NGS. This preliminary action improves the quality and reproducibility of sequencing data while maintaining the high detection capability of NGS for identifying variants.
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 rapid and accurate detection of target nucleic acids, even at low frequencies, facilitating timely and appropriate treatment by simplifying the sample preparation process and improving the sensitivity of genetic analysis.
Implementation Method 1
Since target nucleic acid selectively binds to Cas endonuclease, when bound to the surface of a solid surface, or a particle Cas binds targets in the biological sample to the particle without the need for significant sample preparation
Data Source
AI summary
The invention provides methods for capturing target nucleic acid directly from bodily fluid samples, without the need for certain complex sample preparation steps, using Cas endonuclease to bind to the target nucleic acid sequences. The Cas proteins, along with their sequence-specific guide RNAs, may be introduced directly into the sample, where the Cas proteins bind to ends of a target nucleic acid. The target nucleic acid is thus isolated or enriched in a sequence-specific manner. The target nucleic acid may then be subject to any suitable detection or analysis assay, such as amplification or sequencing. The target nucleic acid may be enriched by digesting other, unbound nucleic acids present in the sample with exonuclease. The bound Cas proteins prevent exonuclease from digesting the target nucleic acid, thereby leaving the only the target nucleic acid substantially present in the sample.