Chromatographic Device for Purifying Double-Stranded Nucleic Acids
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Solution Overview
Problem
Current methods for purifying double-stranded nucleic acids, such as PCR products, are time-consuming, labor-intensive, and require multiple procedural steps, often resulting in incomplete removal of contaminants like single-stranded nucleic acids, mononucleotides, and salts, which is particularly challenging in high-throughput analyses.
Innovation Solution
A chromatographic device with a stationary phase comprising a porous size-exclusion chromatography resin and immobilized metal ions allows for the simultaneous isolation and purification of double-stranded nucleic acids in a single step, effectively removing single-stranded nucleic acids, mononucleotides, and salts by exploiting the differential interactions between immobilized metal ions and the nucleic acid structures.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If alcohol precipitation method is used to purify double-stranded DNA, then DNA can be isolated from reaction mixture, but the method requires multiple time-consuming steps including centrifugation, washing, drying and resolubilization, and yields are dependent on experimenter skill
Solution Approach 1:
The patent combines multiple purification functions (removal of mononucleotides, primers, and salts) into a single chromatography step using a stationary phase with specific binding properties, eliminating the need for separate precipitation, centrifugation, washing, and resolubilization steps required by traditional alcohol precipitation methods
Solution Approach 2:
The patent replaces mechanical operations (centrifugation, manual washing, pipetting) with a chromatographic separation process based on chemical interactions between the stationary phase and nucleic acid structures, enabling automation and reducing experimenter skill dependency
2Reliability
If ExoSAP method is used to process PCR samples, then single-stranded primers and dNTPs can be degraded and dephosphorylated, but salts are not removed and the sample requires additional processing steps
Solution Approach 1:
The patent combines enzymatic treatment (ExoSAP) with chromatographic separation in a single integrated process, where the stationary phase simultaneously removes degraded primers, dephosphorylated dNTPs, and salts through size-exclusion and charge-based mechanisms, eliminating the need for separate salt removal steps
Solution Approach 2:
The stationary phase is designed to perform multiple purification functions simultaneously: removing mononucleotides, degraded primers, salts, and other contaminants in a single pass, making the process universally applicable to various PCR reaction conditions and buffer compositions
3Reliability
If multiple wash steps are performed to remove contaminants, then purification quality improves, but processing time increases and automation becomes difficult
Solution Approach 1:
The patent replaces repetitive mechanical wash steps with a single chromatographic separation process where the stationary phase automatically retains contaminants while allowing purified DNA to pass through, enabling integration into automated liquid handling systems and high-throughput workflows
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 enables rapid and efficient purification of double-stranded nucleic acids with high yield and quality in just two to three steps, avoiding the need for subsequent resolubilization or concentration steps, and minimizes losses and contamination, making it suitable for high-throughput applications.
Implementation Method 1
a stationary phase situated in the cavity, wherein the stationary phase comprises at the least at least one porous chromatography resin, which acts as a size-exclusion chromatography medium
Implementation Method 2
immobilized metal ions... by exploiting the differential interactions between immobilized metal ions and the nucleic acid structures
Data Source
AI summary
The invention relates to a chromatographic device for isolating and/or purifying double-stranded nucleic acids, preferably double-stranded DNA, from a mixture of such nucleic acids with single-stranded nucleic acids, oligonucleotides, mononucleotides, salts and/or other such impurities. The invention also relates to a method for chromatographically isolating and/or purifying same, and to a kit for this purpose.