Pre-treatment Buffer for Nucleic Acid Purification from Clinical Samples
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Solution Overview
Problem
Current nucleic acid extraction and purification methods from clinical samples, especially large samples like stool, are ineffective in removing interfering substances and inhibitors, leading to low-quality nucleic acid preparation that inhibits enzymatic-based analysis.
Innovation Solution
A pre-treatment buffer containing acetate salts and guanidine thiocyanate is used to precipitate and remove interfering substances and inhibitors from clinical samples, followed by centrifugation and subsequent nucleic acid extraction, which results in highly concentrated and purified nucleic acids for analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If conventional nucleic acid extraction methods are used on large clinical samples, then nucleic acids can be extracted, but interfering substances and inhibitors are co-isolated which reduce extraction effectiveness and inhibit enzymatic-based analysis
Solution Approach 1:
The extraction process is divided into distinct segments: first treating the sample with CTAB to precipitate interfering substances, then performing phenol/chloroform extraction to separate nucleic acids from proteins and other contaminants, and finally using alcohol precipitation to isolate pure nucleic acids. This segmentation allows each step to target specific contaminants without co-isolating inhibitors.
Solution Approach 2:
CTAB acts as an intermediary substance that selectively binds to interfering substances and precipitates them, leaving nucleic acids in solution. Phenol and chloroform serve as intermediary solvents that partition contaminants into the organic phase while keeping nucleic acids in the aqueous phase, enabling selective removal of inhibitors.
2Object-affected harmful factors
If prior pre-treatment methods (CTAB, phenol/chloroform extraction, protease treatment) are used, then interfering substances can be reduced, but the protocols are labor intensive with multiple centrifugation and sample transfer steps
Solution Approach 1:
The invention combines CTAB treatment, phenol/chloroform extraction, and protease treatment into a single integrated pre-treatment buffer formulation. This merged buffer performs multiple functions simultaneously: disrupting cell membranes, precipitating interfering substances, denaturing proteins, and preventing inhibitor co-isolation, thereby reducing the number of separate processing steps while maintaining effective contaminant removal.
Solution Approach 2:
The pre-treatment buffer is designed as a universal solution that performs multiple functions: it acts as a cell lysis agent, a precipitating agent for interfering substances, a protein denaturant, and an inhibitor removal agent. This multi-functionality eliminates the need for separate treatment steps for each type of contaminant, simplifying the overall extraction protocol.
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 effectively minimizes the co-isolation of inhibitors, allowing for sufficient nucleic acid loading for enzymatic-based analysis, such as PCR, and simplifies the preparation process by reducing the number of centrifugation steps, particularly in fecal DNA testing for colorectal cancer screening.
Implementation Method 1
a pre-treatment buffer... to precipitate and remove interfering substances and inhibitors from clinical samples
Implementation Method 2
followed by centrifugation and subsequent nucleic acid extraction
Data Source
AI summary
Provided herein is materials and method relating to nucleic acids extraction and purification from biological samples. In particular, a pre-treatment buffer is used to facilitate the extraction and purification of nucleic acids from biological samples, more specifically, removing inhibitors and impurities from biological samples and resulting in a highly concentrated and purified nucleic acids preparation.