Plant Nucleic Acid Isolation Using Chaotropic Lysis and Inhibitor Removal
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Solution Overview
Problem
Nucleic acid isolation from plants is challenging due to the difficult-to-lyse nature of plant cells and the presence of inhibitors, leading to low yields and high inhibitor content in isolated DNA, especially when using methods like mortar and pestle or mechanical disruption with common grinding media.
Innovation Solution
A method involving mechanical disruption with non-spherical solid disrupting particles and a chaotropic agent, followed by protein precipitation and inhibitor removal, to isolate nucleic acids efficiently from various plant samples.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If mortar and pestle or mechanical disruption with common grinding media is used for plant sample lysis, then the lysis process can be performed, but the nucleic acid yield is low and the DNA contains large amounts of inhibitors
Solution Approach 1:
The patent changes the chemical parameters of the lysis buffer by incorporating chaotropic agents (guanidine thiocyanate, guanidine hydrochloride, or guanidine sulfate) at specific concentrations (0.5-2.0 M) to improve cell lysis efficiency and reduce inhibitor content in the isolated nucleic acids
Solution Approach 2:
The patent introduces a chaotropic agent as an intermediary substance that facilitates the lysis process and acts as a mediator to remove inhibitors, enabling effective nucleic acid isolation from plant samples with high inhibitor content
2Productivity
If CTAB lysis buffer is used for lysis and inhibitor removal, then nucleic acids can be isolated, but the CTAB agent is toxic and the method is time-consuming
Solution Approach 1:
The patent replaces CTAB with guanidine-based chaotropic agents that are less toxic and can be easily removed, eliminating the need for complex purification steps and reducing overall processing time while maintaining isolation effectiveness
Solution Approach 2:
The patent substitutes the mechanical mortar and pestle disruption method with a liquid-based lysis system using chaotropic agents, enabling faster, more efficient nucleic acid isolation that can be scaled to multiple samples
3Adaptability or versatility
If standard lysis methods are used for different plant types, then the same protocol can be applied, but the parameters vary dramatically between plant types and parts
Solution Approach 1:
The patent creates a universal lysis protocol using chaotropic agents that can effectively isolate nucleic acids from diverse plant types and parts (leaves, stems, roots, seeds) while maintaining consistent isolation quality through standardized buffer composition and processing conditions
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
The method achieves high-quality nucleic acid isolation with high yield and efficient inhibitor removal, suitable for a wide range of plant types, improving upon existing techniques.
Implementation Method 1
a liquid lysis composition which comprises at least one chaotropic agent
Implementation Method 2
mechanically disrupting the plant sample in a liquid lysis composition which comprises at least one chaotropic agent and one or more solid disrupting particles
Implementation Method 3
contacting the lysed sample with at least one protein precipitating agent
Data Source
AI summary
Provided is a method for isolating nucleic acids from a plant sample comprising(a) preparing a lysed sample wherein preparing comprises(i) lysing a plant sample by mechanically disrupting the plant sample in a lysis solution which comprises at least one chaotropic agent and one or more solid disrupting particles, and(ii) optionally clearing the lysate;(b) contacting the lysed sample with at least one protein precipitating agent and at least one inhibitor removing agent and providing a mixture;(c) obtaining a liquid phase from the mixture; and(d) isolating nucleic acids from the liquid phase.Also provided is a kit for use in such method.