Target Nucleic Acid Extraction From Stool Without Assay Inhibitors
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Solution Overview
Problem
Conventional methods for isolating nucleic acids from complex samples like stool are inadequate for preparing highly concentrated, inhibitor-free DNA from large samples, leading to compromised sensitivity and specificity in diagnostic assays due to the presence of non-target DNA and assay inhibitors.
Innovation Solution
A method involving inhibitor removal using polyvinylpyrrolidone, followed by centrifugation and spin filtration, and direct capture of target nucleic acid using a capture reagent, with optional magnetic separation, to produce a purified nucleic acid solution suitable for diagnostic assays.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If conventional bulk DNA preparation methods are used, then large quantities of DNA can be isolated from large samples, but the DNA preparation contains unacceptable concentrations of assay inhibitors and non-target DNA that compromise assay sensitivity
Solution Approach 1:
The patent divides the bulk DNA preparation process into separate target-specific extraction steps. Instead of isolating all DNA simultaneously, the method uses capture reagents to selectively extract individual target nucleic acids from the sample in sequential steps, thereby separating the desired target DNA from inhibitors and non-target DNA that would be present in conventional bulk preparations
Solution Approach 2:
The patent extracts only the specific target nucleic acid from the complex sample matrix using capture reagents, leaving behind the harmful inhibitors and non-target DNA. This selective extraction approach isolates the useful component (target DNA) while removing the harmful components (inhibitors), resolving the contradiction between obtaining sufficient DNA quantity and eliminating inhibitors
2Measurement precision
If target-specific extraction is performed sequentially, then assay sensitivity is improved by reducing inhibitor concentration, but the extraction process time increases
Solution Approach 1:
The patent performs preliminary removal of major inhibitors from the sample before the sequential target-specific extraction steps. This preliminary cleanup action reduces the inhibitor burden early in the process, allowing subsequent rapid target extractions to proceed with minimal interference, thereby improving sensitivity without proportionally increasing total extraction time
3Measurement precision
If large samples are processed to obtain sufficient DNA, then rare mutations can be detected, but the sample processing complexity and inhibitor removal difficulty increase
Solution Approach 1:
The patent uses capture reagents as intermediary molecules that specifically bind to target nucleic acids in the large sample. These intermediaries facilitate the selective isolation of rare target sequences from the complex matrix, simplifying the processing of large samples by providing a specific binding mechanism that enriches rare targets while removing the bulk of non-target material and inhibitors
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 highly concentrated, inhibitor-free nucleic acid extraction from large samples, enabling sensitive and specific detection of rare mutations and methylation events in diagnostic assays.
Implementation Method 1
removing an assay inhibitor, if present, from the sample to produce a clarified sample
Implementation Method 2
followed by centrifugation and spin filtration
Implementation Method 3
with optional magnetic separation
Data Source
AI summary
Provided herein is technology relating to isolating nucleic acids. In particular, the technology relates to methods and kits for extracting nucleic acids from problematic samples such as stool.