Sequencing-Guided Nucleic Acid Assembly for Long Accurate Synthesis
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Solution Overview
Problem
Existing nucleic acid synthesis techniques are labor-intensive, time-consuming, and prone to errors, especially when synthesizing long polynucleotide chains, and lack efficient quality control for creating entirely synthetic, template-independent molecules.
Innovation Solution
The techniques incorporate quality feedback through sequencing data at various synthesis phases, allowing for the selection and assembly of nucleic acid fragments with desired quality levels, and utilize a single platform for simultaneous synthesis and sequencing to enhance accuracy and efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Length of moving object
If chemical solid phase synthesis is used to form long polynucleotide chains, then the synthesis capability is achieved, but the error rate increases significantly
Solution Approach 1:
The patent divides the synthesis process into two distinct stages: fragment synthesis stage (producing short oligonucleotides with high accuracy) and assembly stage (combining fragments into long polynucleotide chains). This segmentation allows each stage to optimize for its specific function, maintaining high accuracy while achieving long chain synthesis.
Solution Approach 2:
The patent performs preliminary synthesis of multiple short fragments with high accuracy before assembling them into the final long polynucleotide chain. By pre-synthesizing and verifying fragments individually, the overall assembly process benefits from higher starting accuracy and enables error detection and correction before final assembly.
2Adaptability or versatility
If traditional recombinant techniques are used to introduce mutations, then mutation generation is achieved, but labor-intensive analysis and time-consuming host cell propagation are required
Solution Approach 1:
The patent replaces biological host cell propagation and phenotypic analysis with direct in vitro nucleic acid synthesis and sequencing. Instead of using living cells to express and analyze mutations, the invention synthesizes the mutated nucleic acid directly and verifies it through sequencing, eliminating the time-consuming biological propagation step.
Solution Approach 2:
The patent creates direct copies of the desired mutated nucleic acid sequence through chemical synthesis, bypassing the need for biological replication in host cells. The synthesized nucleic acid is then directly sequenced to verify the mutation, replacing the biological copy-making and analysis process with a direct chemical and analytical approach.
3Productivity
If chemical solid phase synthesis is used for short sequences, then synthesis is achieved, but the process becomes slow and error-prone for long sequences
Solution Approach 1:
The patent segments the long polynucleotide synthesis into multiple short fragment syntheses followed by assembly. Each short fragment is synthesized quickly with high accuracy using chemical solid phase synthesis, then multiple fragments are assembled into the final long sequence, combining the speed of short synthesis with the accuracy of modular assembly.
Solution Approach 2:
The patent incorporates sequencing feedback to verify the accuracy of synthesized fragments and assembled polynucleotides. By sequencing the products and comparing them to the intended sequence, the system can detect and correct errors, providing quality control that enables long sequence synthesis while maintaining high accuracy.
Data Source
AI summary
A method for synthesizing a nucleic acid includes synthesizing one or more nucleic acid fragments on a substrate. The synthesized one or more nucleic acid fragments may be amplified on the substrate. The method also includes sequencing the synthesized or amplified one or more nucleic acid fragments on the substrate. The sequencing may provide feedback to designs of the one or more nucleic acid fragments. The method further includes harvesting the synthesized or amplified one or more nucleic acid fragments based on sequencing. The synthesized or amplified one or more nucleic acid fragments may be assembled to generate a target nucleic acid.