Nucleic Acid Isolation Using Phosphate-Chaotropic Lysis
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Solution Overview
Problem
Existing methods for isolating nucleic acids from complex samples like soil and stool samples suffer from low yields and purity due to the presence of contaminating substances and inhibitors, and lack effective lysis methods.
Innovation Solution
A method involving the use of a lytic reagent comprising relatively mild chaotropic agents and phosphates to solubilize nucleic acids, combined with protein-precipitating agents and tri- or tetra-valent salts to remove contaminants, followed by solid phase separation to isolate nucleic acids.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If existing methods are used to isolate nucleic acids from complex samples, then the isolation process can be performed, but the yields and purity are low due to contaminating substances and inhibitors
Solution Approach 1:
The isolation process is divided into distinct stages: lysis with phosphates and chaotropic agents, protein precipitation with salts, and nucleic acid purification. Each stage targets specific contaminants, progressively improving purity while maintaining yield through systematic separation of different contaminant types.
Solution Approach 2:
Phosphates serve as intermediary substances that bind to polyphenolic compounds and tannins, preventing them from interacting with nucleic acids. The chaotropic agents act as intermediaries to denature proteins and disrupt cellular structures, facilitating subsequent separation steps.
2Productivity
If complex samples are lysed using conventional methods, then cell disruption occurs, but the presence of diverse interfering components creates numerous interactions that reduce isolation efficiency
Solution Approach 1:
The method changes chemical parameters by introducing phosphates and chaotropic agents that alter the chemical environment, causing contaminants to precipitate or become separable. This transforms the complex mixture into phases that can be easily separated, improving productivity despite sample complexity.
Solution Approach 2:
The diverse interfering components in complex samples are converted from harmful contaminants into beneficial separation targets. By exploiting the specific chemical properties of different contaminants (polyphenols, proteins, lipids), the method transforms the complexity into a systematic separation opportunity, where each contaminant type responds differently to the reagents.
3Adaptability or versatility
If multiple different biomolecules are isolated from the same sample, then comprehensive analysis is enabled, but the removal of inhibiting components becomes quite challenging
Solution Approach 1:
The phosphate-chaotropic agent system serves multiple functions simultaneously: it precipitates proteins, binds polyphenolics, disrupts lipid membranes, and protects nucleic acids. This universal approach handles diverse contaminants from various biomolecule types (DNA, RNA, proteins) through a single coordinated mechanism, reducing the complexity of inhibitor removal across different isolation protocols.
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 enhances nucleic acid yields and purity while maintaining integrity, allowing for less sample input and facilitating automation and scale-up.
Implementation Method 1
contacting a sample with a lytic reagent comprising one or more phosphates and a chaotropic agent selected from sodium thiocyanate, sodium carbonate, potassium thiocyanate, ammonium thiocyanate, lithium thiocyanate, lithium perchlorate, guanidine sulfate, and combinations thereof to generate a lysate
Implementation Method 2
contacting a sample, a lysate of the sample, or a supernatant of the lysate, or a portion of the sample, the lysate or the supernatant with one or more first agents selected from ammonium acetate, ammonium sulfate, potassium acetate, sodium acetate, sodium chloride, cesium acetate, and combinations thereof, and one or more second agents selected from aluminum chloride, erbium (III) acetate, erbium (III) chloride, holmium chloride, hafnium (IV) chloride, zirconium (IV) chloride, and combinations thereof
Implementation Method 3
separating the mixture of step (a) into a solid phase and a liquid phase, wherein the one or more second agents are primarily in the solid phase
Data Source
AI summary
The present disclosure provides methods for isolating nucleic acids from a sample, comprising: (a) contacting a sample, a lysate of the sample, a supernatant of the lysate, or a portion of the sample, the lysate or the supernatant with one or more first agents (e.g., protein precipitating agents) and one or more second agents (e.g., inhibitor removing agents) to generate a mixture, (b) separating the mixture of step (a) into a solid phase and a liquid phase, wherein the one or more second agents are primarily in the solid phase, and (c) isolating nucleic acids from the liquid phase of step (b). Compositions and kits useful in such methods are also disclosed. Further disclosed are methods, compositions and kits for preparing a lysate using a lytic reagent comprising one or more relatively mild chaotropic agents and one or more phosphates from a sample, especially a complex sample, such as a soil or stool sample.