Linear DNA Multimer Production via Ligation Phase Control
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Solution Overview
Problem
Existing methods for producing DNA molecules with multiple copies of a target sequence often result in intramolecular circularization during ligation, leading to a predominance of circular DNA molecules, which are not suitable for dual beam optical tweezer methods that require linear DNA molecules.
Innovation Solution
A method involving a ligation reaction with a DNA ligase, where the reaction is stopped at the end of the linear phase to prevent further intramolecular circularization, resulting in a mixture of DNA molecules with varying lengths and number of repeats, with a significant portion being linear, by controlling the balance between intermolecular and intramolecular ligation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If a ligation reaction is performed to produce DNA molecules with multiple copies of a target sequence, then the number of repeating units increases, but intramolecular circularization occurs leading to predominance of circular DNA molecules
Solution Approach 1:
The patent applies preliminary action by stopping the ligation reaction at the end of the linear phase, before intramolecular circularization can occur. This timing intervention prevents the formation of circular DNA molecules while still allowing multiple repeating units to be formed through intermolecular ligation. The reaction is quenched at the optimal moment when linear multimeric DNA is maximized but before circularization takes over.
Solution Approach 2:
The patent utilizes the dynamic nature of the ligation reaction by monitoring and controlling the reaction progress through the linear phase. By adjusting reaction conditions and timing, the process dynamically transitions from intermolecular ligation (forming linear multimers) to intramolecular ligation (forming circles), and the invention captures the desired state during this dynamic transition.
2Productivity
If the ligation reaction is allowed to proceed to completion, then all DNA molecules are joined together, but most DNA molecules become circular which are not suitable for optical tweezer methods
Solution Approach 1:
The patent applies partial action by performing the ligation reaction only to the extent needed to form linear multimers with multiple repeating units, rather than allowing complete ligation to occur. The reaction is stopped at the linear phase, providing just enough ligation activity to create the desired multimeric structures without proceeding to the circularization stage, thus achieving partial ligation that is optimal for optical tweezer applications.
3Manufacturing precision
If linear DNA molecules with multiple repeats are produced, then suitable substrates for optical tweezers are obtained, but the production method must control the balance between intermolecular and intramolecular ligation
Solution Approach 1:
The patent applies parameter changes by optimizing ligation reaction conditions such as DNA concentration, ligase amount, and reaction time to control the balance between intermolecular and intramolecular ligation. By adjusting these parameters, the reaction is directed toward forming linear multimers rather than circular molecules, achieving precise control over the molecular architecture suitable for optical tweezer experiments.
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 allows for the production of linear DNA molecules with multiple repeating units, optimizing the length and number of repeats, thereby providing suitable substrates for single-molecule assays like optical tweezers, where circularization is minimized, and linear DNA molecules are predominantly obtained.
Implementation Method 1
subjecting the target DNA sequence to a ligation reaction in the presence of a DNA ligase
Implementation Method 2
Optical tweezers arescientific tools that use a highly focused laser beam to trap and manipulate microscopic or nanoscopic objects
Data Source
AI summary
The invention relates to a method for producing linear DNA molecules with at least two or more repeating units of a target DNA sequence for use as DNA substrate in single-molecule assays, wherein a target DNA is subjected to a ligation reaction and the ligation reaction is stopped when the reaction reaches the end of the linear phase to block circularization of the DNA molecules. Compositions comprising such linear DNA molecules and use of such compositions in single-molecule assays are also provided.