Nucleic Acid Capture Using Elevated Temperature
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Solution Overview
Problem
Current methods for nucleic acid capture using affinity-tagged capture oligonucleotides and immobilized capture agents, such as biotinylated DNA and streptavidin, are inefficient and prone to capturing nonspecific DNA, especially at room temperature.
Innovation Solution
Elevating the temperature during the capture process from 23°C to 60-85°C enhances the speed and efficiency of nucleic acid capture, reducing nonspecific DNA binding and allowing for quicker formation of biotin-streptavidin complexes, while using streptavidin-coated paramagnetic particles immobilized on a solid support.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If room temperature (23°C) is used for nucleic acid capture, then nonspecific DNA binding is reduced, but capture speed and efficiency are slow
Solution Approach 1:
The patent changes the temperature parameter from room temperature (23°C) to elevated temperature (60-85°C) to simultaneously improve capture kinetics and reduce nonspecific binding. This parameter change resolves the contradiction by optimizing both speed and specificity at the same temperature condition.
Solution Approach 2:
The patent introduces dynamic temperature control during the capture process, using elevated temperatures during the capture phase to enhance binding kinetics, then potentially lowering temperature for washing steps to minimize nonspecific binding. This dynamic approach allows optimization of different process stages.
2Loss of time
If room temperature (23°C) is used for nucleic acid capture, then nonspecific binding is minimized, but the time required for capture is long (10-15 minutes)
Solution Approach 1:
The patent applies temperature parameter change to reduce capture time from 10-15 minutes to a shorter duration at elevated temperature (60-85°C), while the high temperature simultaneously reduces nonsspecific binding through increased stringency of the binding interaction.
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 significantly reduces the time required for nucleic acid capture and minimizes the co-extraction of contaminating DNA, improving the yield of target nucleic acid and reducing nonspecific interactions, as demonstrated by experiments showing improved capture efficiency at elevated temperatures.
Implementation Method 1
contacting a sample comprising the target nucleic acid and with an affinity-tagged capture oligonucleotide, wherein the capture oligonucleotide is complementary to all or a portion of the target nucleic acid; incubating the sample under conditions that allow a capture/target complex to form between the capture oligonucleotide and the target nucleic acid
Implementation Method 2
contacting the sample and affinity-tagged capture oligonucleotide with a capture agent, wherein the capture agent and the affinity-tag are capable of forming a stable non-covalent complex; the affinity tag comprises biotin; the capture agent comprises streptavidin
Implementation Method 3
experiments conducted during development of embodiments herein demonstrate that streptavidin capture of biotinylated nucleic acid complexes occurs more quickly and efficiently at elevated temperatures
Implementation Method 4
streptavidin capture of biotinylated nucleic acid complexes occurs more quickly and efficiently at elevated temperatures while simultaneously reducing the amount of nonspecific DNA captured
Implementation Method 5
the capture agent is immobilized on a solid support; the capture agent is a paramagnetic particle (PMP); separating the capture agent from the sample
Data Source
AI summary
Provided herein are methods for enhancing the speed and/or efficiency of a nucleic acid capture using an affinity-tagged capture oligonucleotide (e.g., biotinylated DNA oligo) and an immobilized capture agent (e.g., immobilized avidin/streptavidin). In particular, experiments conducted during development of embodiments herein demonstrate that streptavidin capture of biotinylated nucleic acid complexes occurs more quickly and efficiently at elevated temperatures while simultaneously reducing the amount of nonspecific DNA captured.