Microfluidic Oligonucleotide Assembly for Low-Error Gene Libraries
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Solution Overview
Problem
Existing methods for gene synthesis are limited by scalability, automation, speed, accuracy, and cost, particularly in synthesizing large gene libraries or longer oligonucleotide fragments, with high error rates and a need for more efficient and cost-effective methods.
Innovation Solution
A method for synthesizing n-mer oligonucleotides on a substrate with functionalized loci, coupling building blocks at a specific rate to achieve low error rates, and cleaving the synthesized oligonucleotides, along with a system for conducting parallel reactions using microfluidics to create gene libraries with high fidelity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional gene synthesis methods are used, then synthesis can be performed, but scalability, speed, and cost-effectiveness deteriorate
Solution Approach 1:
The substrate is divided into multiple resolved loci that can be independently addressed, allowing parallel synthesis of multiple oligonucleotides simultaneously. This segmentation enables scalable production while maintaining automated control over each individual synthesis location.
Solution Approach 2:
A functionalized substrate acts as an intermediary platform that holds multiple oligonucleotide chains at resolved loci, enabling parallel processing and automated reagent delivery. The substrate mediates between the synthesis system and the growing oligonucleotide chains, facilitating high-throughput production.
2Reliability
If conventional synthesis methods are used, then synthesis can be performed, but error rate increases
Solution Approach 1:
Chemical synthesis mechanisms replace error-prone biological methods. The chemical coupling reactions at each locus provide precise control over nucleotide incorporation, achieving error rates below 1/500 nucleotides while maintaining high throughput through parallel processing.
Solution Approach 2:
The system incorporates monitoring and control mechanisms that track synthesis progress at each locus, allowing for error detection and correction. This feedback ensures high sequence accuracy while maintaining efficient parallel synthesis across multiple loci.
3Ease of manufacture
If synthesis is performed on substrates with microstructures and clusters, then synthesis can occur, but manufacturing complexity and cost increase
Solution Approach 1:
The substrate provides resolved loci with distinct local properties that enable precise positioning and independent addressing of each synthesis location. This local differentiation allows for high manufacturing precision without requiring complex microstructure fabrication, simplifying the overall manufacturing process.
Data Source
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AI summary
De novo synthesized large libraries of nucleic acids are provided herein with low error rates. Further, devices for the manufacturing of high-quality building blocks, such as oligonucleotides, are described herein. Longer nucleic acids can be synthesized in parallel using microfluidic assemblies. Further, methods herein allow for the fast construction of large libraries of long, high-quality genes. Devices for the manufacturing of large libraries of long and high-quality nucleic acids are further described herein.