One-Pot Multiplex Gene Synthesis via Integrated Phosphorylation and Ligation
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Solution Overview
Problem
Current gene synthesis methods are limited by high costs, low throughput, and the need for error correction and complex protocols, making them difficult to scale and requiring robotic assistance.
Innovation Solution
A method for generating synthetic polynucleotides in a multiplex manner by designing oligonucleotides based on codon usage frequency, performing phosphorylation, ligation, and PCR amplification in a single reaction vessel, allowing for the production of multiple synthetic polynucleotides encoding target proteins.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional gene synthesis assembly methods are used, then gene assembly can be achieved, but errors are generated that require clonal enzymatic correction or sequencing to select error-free assemblies
Solution Approach 1:
The patent combines multiple operations (phosphorylation, ligation, and PCR amplification) into a single one-pot reaction vessel. This integration eliminates the need for separate purification steps between reactions, reducing protocol complexity while maintaining error-free assembly through the coordinated action of all enzymes in the same reaction environment
2Productivity
If conventional gene synthesis methods are used, then gene assembly can be performed, but the methods are difficult to scale-up and involve complicated protocols requiring robotics
Solution Approach 1:
By merging phosphorylation, ligation, and amplification into a single one-pot reaction, the patent enables straightforward scaling without requiring robotic intervention for multiple transfer steps. The simplified protocol can be easily adapted to high-throughput formats using standard laboratory equipment
Solution Approach 2:
The one-pot reaction system uses a universal buffer composition and enzyme mixture that can simultaneously process multiple different oligonucleotide sets targeting different genes. This multi-functionality allows parallel synthesis of multiple genes without requiring separate optimized protocols for each reaction
3Reliability
If conventional gene synthesis methods are used, then gene assembly can be achieved, but the cost is high
Solution Approach 1:
The one-pot reaction eliminates the need for intermediate purification steps, reducing reagent consumption and minimizing material loss. This cost-saving approach maintains high assembly accuracy because the enzymes work cooperatively in the same reaction environment, ensuring faithful oligonucleotide joining without the need for expensive corrective procedures
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 enables cost-effective, error-free, and scalable production of synthetic polynucleotides, reducing the need for robotic protocols and improving the efficiency of gene synthesis, enabling the generation of libraries of proteins.
Implementation Method 1
phosphorylating the plurality of oligonucleotides
Implementation Method 2
performing a ligation reaction with the plurality of phosphorylated oligonucleotides to generate a plurality of nucleic acid templates
Implementation Method 3
performing a PCR reaction in a single reaction vessel to produce the plurality of synthetic polynucleotides
Implementation Method 4
an overhang region forms when the oligonucleotide anneals to another oligonucleotide in the plurality
Data Source
AI summary
The present invention provides methods for generating a library of synthetic polynucleotides. The present invention also provides methods for generating proteins encoded by the library of synthetic polynucleotides. In addition, provided herein are methods for determining the soluble expression of said proteins. This invention is based, in part, on the discovery of a method for selecting optimal oligonucleotides in combination with performing a phosphorylation reaction, ligation reaction and PCR amplification in a single reaction vessel to produce synthetic polynucleotides in a multiplex manner.