Nucleic Acid Isolation Using Low-Salt Lysis and Glass Particle Binding
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Solution Overview
Problem
Current methods for isolating nucleic acids from complex biological samples are time-consuming, hazardous due to the use of chemicals like phenol and chloroform, and require high salt concentrations, which can inhibit downstream applications and damage proteolytic enzymes.
Innovation Solution
A method using a lysis buffer with chaotropic salts and proteolytic enzymes, combined with a binding buffer containing non-chaotropic salts and alcohol, allows for efficient and rapid lysis and binding of nucleic acids to solid phases at lower salt concentrations, reducing the need for extensive washing and minimizing health risks.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If phenol/chloroform extraction is used for nucleic acid isolation, then high purity nucleic acids can be obtained, but health hazards and time consumption increase significantly
Solution Approach 1:
The invention extracts and removes the harmful phenol/chloroform extraction step from the nucleic acid isolation process. Instead, it uses a solid-phase binding approach where nucleic acids are directly bound to glass particles from the lysate, eliminating the need for organic solvent extraction while maintaining high purity results
Solution Approach 2:
The invention replaces reusable but hazardous chemical systems with a disposable solid-phase glass particle system. The glass particles serve as a temporary carrier that can be easily discarded after single use, eliminating ongoing exposure to harmful chemicals while maintaining effective nucleic acid purification
2Productivity
If high salt concentrations are used in lysis buffer for efficient nucleic acid binding, then binding efficiency improves, but proteolytic enzymes are damaged and downstream applications are inhibited
Solution Approach 1:
The invention changes the salt concentration parameter from high (>1.5 M) to low (<0.5 M) in the lysis buffer. This parameter change allows proteolytic enzymes to remain active for effective protein digestion while still enabling efficient nucleic acid binding to the solid phase through alternative mechanisms that do not require high ionic strength
Solution Approach 2:
The invention performs preliminary protein digestion by proteolytic enzymes in the low-salt lysis buffer before nucleic acid binding occurs. This preliminary action ensures proteins are degraded while enzymes remain active, and then nucleic acids are subsequently bound to the solid phase without requiring high salt concentrations that would have denatured the enzymes
3Productivity
If chaotropic salts are used for nucleic acid lysis and binding, then lysis efficiency and binding capacity improve, but extensive washing steps are required to remove salt
Solution Approach 1:
The invention extracts and removes the chaotropic salt component from the lysis/binding buffer system. Instead of using chaotropic salts like guanidine hydrochloride or sodium iodide, the invention employs a low-salt buffer system combined with solid-phase binding, eliminating the need for extensive washing steps to remove high concentrations of chaotropic salts
Solution Approach 2:
The invention introduces a solid-phase glass particle intermediary that enables nucleic acid binding without requiring chaotropic salts. The glass particles serve as a mediator that facilitates direct binding from low-salt lysates, replacing the chaotropic salt intermediary that previously required extensive washing to remove
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 approach enables high-yield, high-purity nucleic acid isolation with reduced labor and costs, while avoiding the inhibitory effects of chaotropic salts on downstream applications.
Implementation Method 1
The lysis buffers cause lysis of the starting material and potent inactivation of nucleolytic enzymes
Implementation Method 2
The formulations include at least one lysis buffer with chaotropic salt components, which additionally contains at least one proteolytic enzyme
Implementation Method 3
binding of the nucleic acids to a solid phase
Implementation Method 4
The binding buffers contain at least one alcohol
Data Source
AI summary
The invention relates to a universal and greatly simplified method of isolating nucleic acids from a wide range of starting materials comprising nucleic acids, in which method a combination of buffers with chaotropic components and buffers with nonchaotropic components is employed.