Size-Exclusion Chromatography for Rapid Nucleic Acid Purification
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Solution Overview
Problem
Current methods for nucleic acid isolation and purification are time-consuming, laborious, and inefficient, often resulting in nucleic acids contaminated with inhibitors that impede amplification and detection, particularly in complex biological samples like blood and tissues.
Innovation Solution
The method employs size-exclusion chromatography to isolate nucleic acids from biological samples using a lysis buffer, followed by amplification and detection, which involves combining the sample with a lysis buffer, subjecting it to size-exclusion chromatography, and then using nucleic acid amplification reagents to amplify the isolated nucleic acids, thereby eliminating inhibitors and simplifying the process.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If classical extraction and washing steps are used for nucleic acid isolation, then purification is achieved, but the process becomes time-consuming and laborious
Solution Approach 1:
The invention extracts only the essential purification function from complex classical methods by using a single size-exclusion chromatography step that selectively separates nucleic acids from inhibitors and cellular debris, eliminating the need for multiple extraction and washing steps while maintaining purification quality
Solution Approach 2:
The invention changes the separation parameter from chemical extraction (phenol, ethanol) to physical size-based separation (size-exclusion chromatography), allowing rapid purification in a single step without the time-consuming multiple steps of classical methods
2Productivity
If magnetic bead-based methods are used for nucleic acid isolation, then isolation speed is improved, but amplification is inhibited by residual agents
Solution Approach 1:
The invention uses disposable size-exclusion chromatography columns that are pre-packed and single-use, eliminating the need for time-consuming washing steps required by magnetic bead methods while ensuring complete removal of inhibitors through the inherent size-based separation mechanism
Solution Approach 2:
The invention employs porous size-exclusion chromatography media that physically separate molecules by size, allowing rapid passage of nucleic acids while retaining larger cellular debris and proteins, achieving both speed and complete inhibitor removal without residual agents
3Reliability
If complex extraction procedures are used, then purification completeness is improved, but operational complexity increases
Solution Approach 1:
The invention segments the purification process into a single integrated size-exclusion chromatography step that simultaneously removes multiple types of inhibitors (proteins, carbohydrates, cellular debris) based on their different sizes, replacing complex multi-step procedures with one simple operation
Solution Approach 2:
The invention creates a universal size-exclusion chromatography method that handles diverse sample types (blood, tissues, biological materials) and removes various inhibitors through a single procedure, making the process equally effective and simple for all applications
4Quantity of substance
If size-exclusion chromatography is used with appropriate volume ratios, then nucleic acid recovery is maximized, but column volume requirements increase
Solution Approach 1:
The invention applies partial action by using a column volume that is only 1-10 times the sample volume (rather than much larger), which is sufficient to achieve the required separation and maximize nucleic acid recovery through the size-exclusion mechanism without excessive column size
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 rapidly isolates nucleic acids in high yields, free from inhibitors, facilitating downstream amplification and analysis, and reduces the need for heat denaturation steps, enhancing the sensitivity of detection for diagnostic applications.
Implementation Method 1
Size-exclusion chromatography (SEC), also called gel-filtration or gel-permeation chromatography (GPC), uses porous particles stacked within a column to separate molecules of different sizes
Implementation Method 2
Size-exclusion chromatography (SEC), also called gel-filtration or gel-permeation chromatography (GPC), uses porous particles stacked within a column to separate molecules of different sizes
Implementation Method 3
combining the biological sample with a lysis buffer to form a lysis mixture comprising nucleic acid released from cells in said biological sample
Data Source
AI summary
The invention provides methods of detecting a nucleic acid present in a biological sample, comprising combining the biological sample with a lysis buffer to form a lysis mixture comprising nucleic acid released from cells in said biological sample; and subjecting a volume of the lysis mixture to size-exclusion chromatography in a column comprising a volume of size-exclusion medium. In certain embodiments, the lysis buffer separates double-stranded nucleic acid into single-stranded nucleic acid. In certain embodiments, the elution can have a flow rate of separation of less than 10 minutes to produce an eluted solution comprising isolated nucleic acid. The invention provides for a method of accurately and rapidly detecting products of nucleic acid amplification.