Silicate Filter Microbial DNA Isolation
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Solution Overview
Problem
Current methods for DNA analysis from microbial samples are hindered by the inability to distinguish between live and dead cells, as biofilms complicate filtration and centrifugation, leading to erroneous DNA assessments and the need for time-consuming purification steps to remove interfering dyes.
Innovation Solution
A method utilizing a macroporous silicate filter to capture intact cells and cell-free nucleic acids, where nuclease degradation and elution buffers are used to isolate and purify DNA from intact cells, separating it from dead cell DNA and biofilm contaminants.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If filtration or centrifugation is used to collect microbes, then microbial DNA can be obtained, but biofilm and dead cells contaminate the sample leading to erroneous DNA assessments
Solution Approach 1:
The patent extracts and removes dead cells and biofilm contaminants from the microbial sample using selective lysis methods and filtration, isolating only intact live cells for DNA analysis. This extraction of harmful components resolves the contradiction by ensuring DNA analysis accuracy while maintaining reliability of live cell differentiation.
Solution Approach 2:
The patent applies preliminary action by treating the sample with nucleases and lysis buffers before DNA extraction to pre-differentiate and remove dead cells and biofilm. This preliminary processing step ensures that only intact cells with protective membranes remain, eliminating contamination sources before the actual DNA analysis, thus improving both measurement precision and reliability.
2Measurement precision
If propidium azide dye is used to distinguish live and dead cells, then live cell identification is achieved, but additional purification steps are required to remove interfering dyes
Solution Approach 1:
The patent takes out and removes the propidium azide dye and other interfering substances from the sample through selective lysis and purification steps. By extracting these harmful components, the method achieves accurate live cell identification without requiring additional time-consuming purification steps, thus resolving the contradiction between measurement precision and time loss.
Solution Approach 2:
The patent applies preliminary action by using nucleases and lysis buffers to pre-treat the sample before DNA extraction, which pre-removes interfering dyes and contaminants. This preliminary processing eliminates the need for subsequent purification steps, achieving both accurate live cell identification and time efficiency.
3Productivity
If conventional filtration methods are used, then microbial collection is achieved, but biofilm patches contaminate the sample making DNA analysis erroneous
Solution Approach 1:
The patent extracts and removes biofilm patches and dead cells from the microbial collection using selective lysis methods and filtration with specific pore sizes. This extraction of contaminants allows the method to maintain high microbial collection efficiency while ensuring DNA analysis accuracy, resolving the contradiction between productivity and measurement precision.
Solution Approach 2:
The patent applies preliminary action by pre-treating the sample with nucleases and lysis buffers before filtration to break down biofilm structures and differentiate live from dead cells. This preliminary processing ensures that only intact live cells pass through the filter, maintaining collection efficiency while improving DNA analysis accuracy.
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 effectively isolates DNA from intact cells while eliminating dead cell DNA and biofilm contaminants, enabling accurate DNA analysis without the need for additional purification steps, thus improving the reliability of microbial DNA assessments.
Implementation Method 1
The intact cells and the cell free nucleic acids are captured within a matrix comprising a macroporous filter
Implementation Method 2
The captured cell free nucleic acids are degraded in situ with a nuclease
Implementation Method 3
Cellular nucleic acids are released from the captured intact cells and eluted from the matrix with an elution buffer
Data Source
AI summary
Provided herein are methods for isolating nucleic acids from intact cells in a sample of intact cells and cell free nucleic acids. The intact cells and cell free nucleic acids are captured and concentrated within the pores of a silicate matrix in a microporous silicate filter. Within the silicate matrix the cell free nucleic acids are degraded with a nuclease, the intact cells are lysed with the released DNA binding to the silicate, the nuclease treated cell free nucleic acids and contaminants from the lysed are washed from the silicate matrix and the DNA bound to the silicate is eluted therefrom to form an isolated DNA product.