Nucleic Acid Isolation via Chaotropic Salt Buffer
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Solution Overview
Problem
Current methods for isolating nucleic acid fragments of specific sizes in molecular diagnosis, such as next-generation sequencing, face challenges including time-consuming processes, inferior resolution, and increased costs due to the need for specific modified magnetic beads or pH adjustments, which limit their applicability and accuracy.
Innovation Solution
A method and kit using a simple reaction buffer solution with chaotropic salts and monovalent salts or crowding agents to selectively isolate nucleic acid fragments of specific sizes without relying on pH adjustments or modified functional groups, suitable for automation systems and high-throughput operations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If agarose gel electrophoresis is used to separate nucleic acid fragments by size, then the target fragments can be recovered accurately, but the process is time-consuming
Solution Approach 1:
The patent replaces the mechanical electrophoresis separation system with a chemical binding system. Magnetic beads coated with specific DNA sequences selectively bind to target nucleic acid fragments through hybridization, allowing size-based separation without electrical fields or gels. This substitution dramatically reduces processing time while maintaining recovery accuracy.
Solution Approach 2:
The patent introduces magnetic beads coated with conjugating sequences as an intermediary between the nucleic acid sample and the detection system. These beads act as selective mediators that bind specifically to target fragments of desired sizes, enabling accurate recovery without the time-consuming electrophoresis process.
2Extent of automation
If magnetic beads with DNA molecular coating and surface functional group modification are used, then automation is improved, but additional costs and processes are incurred
Solution Approach 1:
The patent changes the binding mechanism parameter from chemical functional group interactions to nucleic acid hybridization. By using magnetic beads coated with DNA sequences that hybridize to complementary sequences on target fragments, the system achieves automation without requiring complex surface chemistry modifications or additional processing steps for functional group activation.
Solution Approach 2:
The patent creates a universal binding system where magnetic beads with conjugating sequences can bind to any nucleic acid fragment containing the complementary sequence, regardless of fragment size. This multi-functional approach allows a single bead type to handle various target sizes, reducing the need for multiple specialized reagents and simplifying the overall process.
3Reliability
If modified magnetic beads are used for isolating small-size fragments, then recovery effect is improved, but application range is limited to fragments smaller than 400 bp
Solution Approach 1:
The patent introduces dynamic control over fragment size selection through adjustable parameters. By modifying the size distribution of the nucleic acid sample before hybridization or adjusting hybridization conditions, the same magnetic bead system can selectively recover fragments of different sizes, making the method adaptable to both small fragments (＜400 bp) and larger fragments while maintaining high recovery effectiveness.
4Measurement precision
If primer or bait is used to recover nucleic acid, then accuracy is improved, but the method is difficult to use widely
Solution Approach 1:
The patent creates a universal recovery system using magnetic beads coated with conjugating sequences that can bind to any nucleic acid fragment containing the complementary sequence. Unlike primers or baits that require specific sequence knowledge, this system works with any fragment size or sequence composition, dramatically improving applicability while maintaining accuracy through selective hybridization.
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 efficient and cost-effective isolation of nucleic acid fragments of specific sizes, improving the quality and quantity of sequencing results while maintaining a stable solution environment, suitable for a wide range of nucleic acid sizes and applications.
Implementation Method 1
the mixing solution has a chaotropic salt and one or two of a monovalent salt or a crowding agent
Implementation Method 2
when the concentration of monovalent salt is high, the large-size nucleic acid fragment is bound to a solid carrier and when the concentration of monovalent salt is low, the bigger range (includes large-size and small size) of nucleic acid fragments is bound to the solid carrier
Data Source
Figure 1A~1C
Figure 2A
Figure 2B
AI summary
The present invention provides a method and a kit for selective isolation of nucleic acid. The selection or filtration of nucleic acid fragments is achieved by binding the a solid carrier with nucleic acid fragments in different sizes, with the help of the association of chaotropic salts, monovalent salts and crowding agents in different types and concentrations.