Organic Solvent Extraction Buffer for Nucleic Acid Inhibitor Removal
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Solution Overview
Problem
Current nucleic acid extraction methods are labor-intensive, costly, and often require boiling, which is not safe or convenient in clinical settings, leading to challenges in obtaining high-quality nucleic acids free from inhibitors for downstream applications like PCR and isothermal amplification.
Innovation Solution
A method using low concentrations of organic solvents such as acetone or ethanol in an extraction buffer, combined with short incubation at lower temperatures, to effectively remove inhibitors and extract nucleic acids without the need for boiling, allowing for simpler and safer sample preparation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional nucleic acid extraction methods are used, then nucleic acids can be extracted, but the process is labor-intensive and time-consuming
Solution Approach 1:
The invention changes the chemical parameters of the extraction buffer by adding organic solvents (ethanol, acetone, or butanol) at specific concentrations (3-20% v/v). This parameter change enables rapid inhibitor removal and simplifies the extraction process, reducing it from multiple lengthy steps to a single incubation step of 5-30 minutes, thereby dramatically improving productivity while minimizing time loss.
2Ease of operation
If boiling is used for crude nucleic acid preparation, then nucleic acids can be extracted, but it is unsafe and inconvenient in clinical settings
Solution Approach 1:
The invention changes the temperature parameter from high-temperature boiling to lower temperature incubation (15-35°C for 5-30 minutes) in the presence of organic solvents. This parameter change maintains nucleic acid extraction effectiveness while eliminating the safety hazards and inconvenience of boiling, making the procedure suitable for clinical settings without compromising reliability.
Solution Approach 2:
The invention introduces organic solvents (ethanol, acetone, or butanol) as intermediary substances that facilitate inhibitor removal and nucleic acid extraction at lower temperatures. These intermediaries enable the process to achieve reliable nucleic acid preparation without requiring dangerous boiling conditions, thus improving ease of operation while maintaining reliability.
3Reliability
If extensive sample processing is performed, then nucleic acid quality improves, but the complexity of the procedure increases
Solution Approach 1:
The invention merges multiple separate steps (enzymatic digestion, incubation, separation, precipitation, elution) into a single integrated incubation step in the presence of organic solvents. This merging maintains nucleic acid quality by effectively removing inhibitors while dramatically reducing procedure complexity from multiple sequential operations to one simple incubation followed by direct amplification.
Solution Approach 2:
The invention changes the chemical composition parameter of the extraction buffer by incorporating organic solvents at optimized concentrations. This parameter change enables the buffer to simultaneously perform multiple functions (lysis, inhibitor removal, and nucleic acid stabilization) that previously required separate processing steps, thereby improving nucleic acid quality while simplifying the overall procedure.
4Reliability
If conventional extraction buffers without organic solvents are used, then the procedure is simpler, but amplification inhibitors remain in the extract
Solution Approach 1:
The invention changes the compositional parameters of the extraction buffer by adding organic solvents (ethanol, acetone, or butanol) at specific concentrations (3-20% v/v). This parameter change enables the buffer to effectively remove amplification inhibitors while maintaining procedural simplicity, as the organic solvents are directly incorporated into the buffer without requiring additional reagents or complex preparation 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 reduces or eliminates amplification inhibitors, enabling successful nucleic acid amplification with high-quality DNA and RNA extracts suitable for various molecular diagnostics, including PCR and isothermal amplification, while being safer and more cost-effective than traditional methods.
Implementation Method 1
diluting a biological sample in an extraction buffer to form a mixture, wherein the extraction buffer comprises 3 wt. % to 10 wt. % organic solvent... incubating the mixture at a temperature from 15°C to 35°C for five seconds to thirty minutes, to cause nucleic acid present in the biological sample to be extracted
Implementation Method 2
separating at least a portion of the extraction buffer comprising at least a portion of the nucleic acid from the biological sample by heating for 3-5 minutes at 65°C
Data Source
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AI summary
Nucleic acid amplification tests have been widely used in clinical laboratories. Nucleic acid extraction from biological materials is challenging because different unfavorable substances may co-extract and inhibit downstream applications. The present claimed invention relates to a composition of and a method for treating the sample prior, during or post extraction of nucleic acid. More specifically, the claimed invention relates to a composition of and a method for using low concentrations of common organic solvents to remove inhibitors of nucleic acid amplification. The present claimed invention can be used for extracting nucleic acids (DNA/RNA) from bacteria, viruses, parasites, and other biological materials or matrices, including but not limit to, stool samples, body fluids, plants and cultures. The method is rapid, low-cost, and easy to use in a laboratory setting. The nucleic acid extracted in accordance with the claimed invention can be used for nucleic acid amplification reactions.