Transposase Nucleic Acid Fragmentation Without Purification
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Solution Overview
Problem
Current nucleic acid fragmentation methods using transposase require additional purification steps and increase costs when dealing with higher starting amounts of DNA, as transposase must be removed using column or magnetic beads, complicating the process.
Innovation Solution
A one-stop treatment method and reagent system that uses a transposase-embedded complex with specific reagents to break the adsorption effect of transposase on nucleic acids, allowing for direct PCR amplification without the need for purification, utilizing SDS, protease, EDTA, Triton-X100, and Tween-20 to manage transposase interference and ensure smooth enzymatic reactions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If transposase is used for nucleic acid fragmentation with higher starting amounts of DNA, then fragmentation efficiency is improved, but additional purification steps are required which increases process complexity and costs
Solution Approach 1:
The patent combines the transposase fragmentation step and the purification step into a single integrated reagent system. The reagent contains transposase, SDS, protease, EDTA, and buffer components that work together in one tube to both fragment the nucleic acid and remove the transposase protein, eliminating the need for separate purification operations.
Solution Approach 2:
The reagent system performs multiple functions simultaneously: it fragments nucleic acid via transposase activity, denatures the transposase protein via SDS, degrades the protein via protease, and chelates metal ions via EDTA. This multi-functional reagent replaces what would traditionally require multiple separate reagents and steps.
2Reliability
If column or magnetic beads purification is used to remove transposase, then transposase interference in downstream reactions is eliminated, but experimental costs and time consumption increase
Solution Approach 1:
The reagent system maintains continuous action throughout the reaction process. The transposase works to fragment DNA, and simultaneously the other components (SDS, protease, EDTA) work to remove the transposase. This continuous parallel processing eliminates idle purification steps and maintains workflow continuity.
Solution Approach 2:
The system is self-cleaning - the transposase fragmentation reaction generates the need for purification, and the same reaction mixture contains all components needed to perform that purification. The reagent serves itself by incorporating both the fragmentation mechanism and the cleanup mechanism in a single system.
3Quantity of substance
If transposase amount is increased for higher starting DNA amounts, then fragmentation coverage is improved, but the need for purification becomes mandatory which complicates the procedure
Solution Approach 1:
The patent changes the chemical parameters of the reaction environment by adding SDS (detergent), protease (enzyme), and EDTA (chelating agent). These parameter changes transform the system from one where transposase remains bound to DNA to one where transposase is denatured and removed, allowing the system to handle higher transposase amounts without purification 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
Simplifies the experimental procedure, reduces costs, and enables high-throughput sample processing by eliminating the need for column or magnetic beads purification, allowing for seamless transition from nucleic acid interruption to downstream PCR amplification.
Implementation Method 1
adding a first reagent for treatment, so as to break the adsorption effect of the transposase and the target sequence of the nucleic acid
Implementation Method 2
the first reagent comprises a sodium dodecyl sulfate (SDS) solution and a NT buffer... or a protease solution
Implementation Method 3
EDTA, whose chemical name ethylenediaminetetraacetic acid, is further added for treatment after the treatment with the first reagent
Implementation Method 4
the second reagent comprises Triton-X100 solution. Triton-X100, whose chemical name octylphenyl polyoxyethylene ether, as a nonionic surfactant, in the role of the present invention is to weaken the influence of the first reagent on the subsequent enzymatic reactions
Implementation Method 5
the second reagent further comprises a Tween-20... Tween-20 may be used as a component of the second reagent in the form of a mixture with Triton-XlOO
Data Source
Figure 1A~2
Figure 3
AI summary
Disclosed are a one-stop treatment method for breaking a nucleic acid by means of a transposase, and a reagent. The method of the present invention comprises the following steps: conducting random breaking of a nucleic acid by using a transposase-embedded complex, the transposase-embedded complex comprising a transposase and a first adaptor comprising a transposase identification sequence; adding a first reagent to conduct treatment, so as to break an absorption effect of the transposase to a target sequence of the nucleic acid; adding a second reagent to conduct treatment, so as to weaken the influence of the first reagent on a follow-up enzyme-catalyzed reaction; and conducting a PCR reaction by using a product generated after the second reagent treatment as a template component, so as to obtain a PCR product of a broken nucleic acid segment whose two ends are connected to adaptors.