Nucleic Acid Isolation via DMB Precipitation
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for isolating nucleic acids from extracellular vesicles (EVs) face challenges such as inefficiency in small sample volumes, damage from high-speed centrifugation, and contamination issues, which hinder their use in diagnostics and personalized medicine applications.
Innovation Solution
A method involving dimethylmethylene blue (DMB) dye at a pH between 2 and 6.9, which precipitates EVs, allowing for the efficient isolation of nucleic acids without prior enrichment, reducing contaminant proteins, and maintaining EV integrity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If differential ultracentrifugation or density gradient ultracentrifugation is used for EVs purification, then EVs can be separated from biofluids, but the integrity of EVs may be damaged due to prolonged high speed ultracentrifugation and membrane debris are observed
Solution Approach 1:
The patent replaces the mechanical ultracentrifugation system with a chemical precipitation system using PEG. Instead of using high-speed centrifugal force to separate EVs, the method uses PEG to induce precipitation of EVs from biofluids through chemical interactions, thereby avoiding mechanical damage to EV membranes while achieving efficient separation and recovery.
2Reliability
If size exclusion chromatography is used for EVs isolation, then EVs can be separated without high speed centrifugation, but EVs may be lost by binding to membranes and deformation may occur
Solution Approach 1:
The patent replaces the mechanical chromatography separation system with a chemical precipitation system using PEG. This substitution avoids the need for EVs to pass through chromatography membranes, preventing binding losses and deformation, while still achieving effective EV isolation through controlled precipitation and subsequent low-speed centrifugation.
3Measurement precision
If conventional EVs isolation methods are used, then EVs can be purified, but the recovery of exosomal RNA and proteins is not optimal and contaminant proteins remain
Solution Approach 1:
The patent optimizes specific parameters of the PEG precipitation process, including PEG concentration (8-15% w/v), pH (6.5-7.5), ionic strength (150-250 mM NaCl), and incubation temperature (4-37°C), to achieve selective precipitation of EVs while minimizing co-precipitation of contaminant proteins and maximizing the recovery of associated nucleic acids and proteins.
4Volume of moving object
If small volumes of biological samples are used for EVs isolation, then sample volume is reduced, but the efficiency in recovery of sufficient amounts of EVs is limited
Solution Approach 1:
The patent optimizes PEG precipitation parameters specifically for small volume samples, adjusting PEG concentration, incubation time, and temperature to maximize EV recovery efficiency from limited sample volumes, thereby enabling effective EV isolation even from microliter-scale biofluids without requiring sample concentration steps.
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 enables the recovery of sufficient nucleic acids from small sample volumes, improving the efficiency and purity of EV isolation, making it suitable for diagnostic, prognostic, and therapeutic applications, including personalized medicine.
Implementation Method 1
contacting the sample with the dimethylmethylene blue (DMB) dye at a pH comprised between 2 and 6.9; incubating the mixture from a) at a temperature comprised between 0° C. and 40° C. for the time required for the formation of a DMB-EVs precipitate
Data Source
AI summary
The invention relates to an in vitro method for isolating nucleic acids associated to or contained inside extracellular vesicles (EVs) from a sample based on the formation of a DMB-EVs precipitate and the isolation of the nucleic acids present in the precipitate. The invention also relates to the use of the method of the invention for diagnosing or for determining the susceptibility of a subject to a disease, for determining the prognosis or for monitoring the progression of a disease, for monitoring the effect of a therapy, for identifying compounds suitable for the treatment of a disease, or for designing a personalized therapy or selecting a patient susceptible to being treated with a therapy for the prevention and/or treatment of a disease. In addition, the invention also relates to a kit comprising dimethylmethylene blue (DMB) and a reagent capable of isolating nucleic acids from EVs, and to its use.