Transient Drag-Tags for DNA Sequencing Read Lengths
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Solution Overview
Problem
Current DNA sequencing methodologies, such as capillary gel electrophoresis, are limited by diffusion band broadening and require long run times, while end labeled free solution electrophoresis (ELFSE) is restricted by the lack of large, monodispersed polymeric drag-tags, which limits achievable sequencing read lengths.
Innovation Solution
The method involves transiently attaching drag-tags to DNA fragments instead of covalently bonding polydispersed polymers, using a running buffer with surfactant micelles or other structures to enhance separation efficiency by reducing band broadening and increasing sequencing read lengths.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If covalently attaching polydispersed polymers to DNA fragments is used, then drag-tags can be provided to enable separation, but the polydispersity causes band broadening and limits sequencing read lengths
Solution Approach 1:
The patent applies the dynamics principle by transitioning from static covalent bonding to dynamic transient interactions. The drag-tag system uses reversible non-covalent binding (e.g., hydrophobic interactions, host-guest chemistry) that allows continuous association and dissociation during electrophoresis. This dynamic exchange ensures that even with polydispersed drag-tags, the instantaneous interactions maintain consistent effective drag forces, eliminating band broadening caused by polydispersity while preserving separation resolution.
2Measurement precision
If capillary gel electrophoresis is used for DNA sequencing, then separation can be achieved, but long run times are required which cause diffusion band broadening
Solution Approach 1:
The patent replaces the mechanical gel matrix system with a free-solution electrophoresis system. Instead of using a physical gel matrix that requires long run times and causes diffusion broadening, the invention uses drag-tags with transient interactions to provide the necessary frictional drag in free solution. This substitution allows for much shorter run times (reducing diffusion effects) while maintaining separation resolution through the controlled transient drag forces.
3Productivity
If ELFSE with polydispersed drag-tags is used, then separation can occur, but the lack of monodispersed polymeric drag-tags limits achievable sequencing read lengths
Solution Approach 1:
The patent introduces an intermediary binding mechanism (transient non-covalent interactions) between the drag-tag and DNA fragment. This intermediary role allows polydispersed drag-tags to function effectively because the transient binding ensures that at any given moment, the DNA experiences a consistent effective drag force regardless of the full distribution of drag-tag sizes. The intermediary interaction mechanism decouples the polydispersity of the drag-tag population from the separation performance, enabling long sequencing read lengths.
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 higher sequencing read lengths and improved separation performance by mitigating the effects of drag-tag polydispersity, enabling longer DNA fragment separation without significant loss of resolution.
Implementation Method 1
the lipophilic moiety hydrophobically interacts with the drag-tag
Implementation Method 2
the molecule moves by the electrostatic force between the polar moiety and the electric field
Implementation Method 3
Thermal motion or an electrostatic force eventually breaks the hydrophobic interaction
Data Source
AI summary
The invention relates transiently attaching drag-tags to molecules during electrophoresis. The invention includes running buffers having drag-tags that transiently attach to lipophilic moieties attached to the molecules. The lipophilic moieties can be covalently or ionically bonded to the molecules. One particular aspect of the invention is a nucleoside analog or a nucleic acid analog comprising a lipophilic moiety. The invention is also directed to methods of separating molecules that comprise a lipophilic moiety. The methods generally comprise transiently attaching a drag-tag to the lipophilic moiety during a separation modality. These methods can be used to separate the molecules by size or weight, to measure a hydrodynamic radius of a drag-tag, or to separate a plurality of drag-tag by their hydrodynamic radius.