Double-Layer Chromatography Column for Nucleic Acid Isolation
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Solution Overview
Problem
Current nucleic acid isolation methods, such as silica-based and anion exchange-based methods, face limitations in efficiently isolating nucleic acids from large volumes like plasma and in co-isolating RNA and DNA, especially when they are bound to proteins, and require additional steps to prepare the isolated nucleic acids for downstream applications.
Innovation Solution
A double-layer chromatography method using a first anion exchange layer and a second silica layer, where nucleic acids are bound and eluted in a sequential process with specific salt and pH conditions, allowing for efficient co-isolation and direct readiness for downstream applications.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If silica-based isolation method is used, then nucleic acid binding efficiency is improved, but the starting volume of samples is limited due to the need to add large volumes of chaotropic salt solution
Solution Approach 1:
The method segments the isolation process into two distinct stages: first capturing nucleic acids from large sample volumes using anion exchange at low pH, then transferring and concentrating them onto silica for final purification. This segmentation allows each stage to optimize for its specific function without the limitations of using a single method for the entire process.
Solution Approach 2:
The anion exchange resin acts as an intermediary medium that temporarily captures nucleic acids from large sample volumes, enabling concentration before transfer to silica. This intermediary step allows the system to handle large starting volumes that would otherwise be incompatible with silica-based methods.
2Ease of manufacture
If conventional isolation methods are used, then nucleic acid isolation can be performed, but additional steps are required to prepare isolated nucleic acids for downstream applications
Solution Approach 1:
The method merges the advantages of anion exchange (handling large volumes, co-isolating RNA and DNA) with silica-based purification (high purity, downstream readiness) into a single integrated two-layer column system. This combination eliminates the need for separate preparation steps while maintaining simplicity.
Solution Approach 2:
The dual-layer column design provides multi-functionality: the anion exchange layer handles sample loading and initial concentration from large volumes, while the silica layer simultaneously provides purification and prepares nucleic acids for downstream applications. This universal system handles multiple functions in one operation.
3Device complexity
If single-layer chromatography is used, then device complexity is reduced, but the ability to co-isolate RNA and DNA from large volumes bound to proteins is limited
Solution Approach 1:
The chromatography column uses composite materials by combining anion exchange resin and silica particles in a two-layer structure. Each material contributes its unique properties: anion exchange for capturing nucleic acids from complex matrices, and silica for purification and concentration. This composite approach enables co-isolation capabilities that neither material could achieve alone.
Solution Approach 2:
The invention adds a vertical dimension to the chromatography system by stacking two functional layers, rather than using a single layer. This dimensional approach allows sequential processing of different sample types and conditions within one column, enhancing versatility without proportionally increasing horizontal complexity.
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 isolates nucleic acids from large volumes and those bound to proteins, ensuring high purity and readiness for downstream applications without the need for additional purification steps, as demonstrated by successful DNA recovery and quality in agarose gel electrophoresis and real-time PCR assays.
Implementation Method 1
a first anion exchange layer... nucleic acids are bound and eluted in a sequential process
Implementation Method 2
The binding principle is based on the interaction between the negatively charged phosphates of the DNA backbone and the positively charged group
Implementation Method 3
The principle of silica-based isolation is based on the high affinity of the negatively charged DNA backbone towards the positively charged silica surface under concentrated chaotrophic salt conditions
Implementation Method 4
GuTC and GuHCl are commonly used for binding nucleic acid to the silica surface... guanidinium salts, e.g., GuTC, are known to efficiently lyse cells and denature proteins
Implementation Method 5
DNA is eluted in the presence of a high salt concentration at a high pH value
Implementation Method 6
DNA is eluted in the presence of a high salt concentration
Data Source
AI summary
The present invention provides a chromatography column and a method for isolating nucleic acid molecules. In one embodiment, the present invention provides a double-layer column of a first anion exchange membrane and a second serially coupled silica membrane. Upon flowing a nucleic acid-containing solution through the first anion exchange membrane, the nucleic acid binds to and then elutes from the first membrane. The eluted solution then flows serially through the second silica membrane, which the nucleic acid binds to and then elutes from. Due to this novel serial coupled double-layer principle, the present invention is particularly suitable for co-isolating RNA and DNA, for isolating nucleic acid embraced by proteins, e.g., viruses, and for isolating diluted nucleic acid in a large volume, e.g., plasma. In addition, the eluted nucleic acid is ready for downstream applications.