On-array ligation assembly for DNA construct synthesis
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Solution Overview
Problem
Current high-throughput methods for DNA construct assembly are labor-intensive and costly due to the high reagent requirements for joining shorter oligonucleotides into longer constructs, despite advancements in polymerase or ligase enzyme-based methods.
Innovation Solution
A method involving hybridization of double-stranded oligonucleotides to a substrate with surface-tethered oligonucleotides, followed by ligation of surface-distal ends to produce a ligation product tethered to the support at both ends, allowing for efficient assembly and subsequent cleavage to extend the construct.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Extent of automation
If automated robotic systems with micro-titer plates are used for DNA assembly, then labor costs are reduced, but reagent costs and complexity increase due to multiple reactions required
Solution Approach 1:
The DNA assembly process is segmented into two distinct phases: (1) hybridization of double-stranded oligonucleotides to surface-tethered oligonucleotides on a solid support, and (2) ligation of the surface-distal ends. This segmentation allows the oligonucleotides to be immobilized during assembly, enabling washing away of excess reagents and reducing the quantity of reagents needed compared to traditional solution-based methods that require multiple sequential reactions in micro-titer plates.
Solution Approach 2:
The invention transitions from traditional two-dimensional micro-titer plate-based assembly to a three-dimensional solid support system where oligonucleotides are tethered to a surface. This dimensional change allows for efficient immobilization and reduces reagent consumption by confining the reaction to the surface-bound molecules rather than requiring excess reagents in solution phase.
2Adaptability or versatility
If traditional oligonucleotide assembly methods are used, then flexibility in method selection exists, but labor intensity and reagent consumption increase
Solution Approach 1:
The solid support performs a self-service function by automatically immobilizing the hybridized double-stranded oligonucleotides during the hybridization step. This self-immobilization eliminates the need for manual transfer operations and reduces labor intensity. The surface-tethered oligonucleotides serve as both the substrate and the anchoring mechanism, streamlining the process while maintaining versatility in oligonucleotide sequence selection.
3Reliability
If multiple sequential reactions are performed in micro-titer plates, then complete assembly can be achieved, but process complexity and reagent volume increase
Solution Approach 1:
The invention merges the hybridization and ligation steps into a streamlined two-step process on a solid support. The surface-tethered oligonucleotides remain stationary during both steps, allowing excess reagents to be washed away between steps without requiring complex liquid handling operations. This merging of steps on a fixed support reduces process complexity compared to performing multiple sequential reactions in micro-titer plates while ensuring complete assembly through thorough washing and controlled reaction conditions.
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 reduces reagent costs and labor by enabling efficient assembly of DNA constructs on a support, facilitating the production of longer DNA sequences while minimizing the need for extensive reagent usage and optimizing the assembly process.
Implementation Method 1
hybridizing a first double-stranded oligonucleotides and a second double-stranded oligonucleotide to a substrate comprising surface-tethered oligonucleotides
Implementation Method 2
ligating the surface-distal ends of the first and second double-stranded oligonucleotides together, thereby producing a first ligation product
Data Source
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AI summary
Provided herein, among other things, is a method for producing a ligation product on a support. In some embodiments, the method may comprise hybridizing a first double-stranded oligonucleotides and a second double-stranded oligonucleotide to a substrate comprising surface-tethered oligonucleotides and ligating the distal ends of the first and second double-stranded oligonucleotides together, thereby producing a first ligation product that is tethered to the support at both ends.