Seamless Nucleic Acid Assembly via Bacterial Lysate Recombination
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Solution Overview
Problem
Current methods for nucleic acid synthesis face challenges with scalability, automation, speed, accuracy, and cost, particularly in assembling long sequences efficiently and accurately.
Innovation Solution
A method involving the use of double-stranded nucleic acids with specific adapter sequences and homology regions, mixed with a bacterial lysate containing nuclease or recombinase, to facilitate seamless assembly through processes like in vitro recombination cloning and single-stranded DNA mediated hierarchal assembly, achieving high percentages of correct assembly.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If traditional nucleic acid synthesis methods are used, then short fragments can be synthesized, but scalability and assembly accuracy deteriorate
Solution Approach 1:
The patent divides long nucleic acid sequences into multiple overlapping short fragments that can be independently synthesized with high accuracy, then assembled together using homology-based recombination. This segmentation allows each fragment to be synthesized with high precision while the overall system achieves scalability through parallel processing of multiple fragments.
Solution Approach 2:
The patent introduces homology sequences as intermediary elements that mediate the assembly between synthesized fragments and the target vector. These homology sequences serve as recognition and binding sites that facilitate accurate joining without requiring traditional cloning methods, thereby improving both assembly accuracy and scalability.
2Productivity
If assembly methods are simplified for scalability, then productivity improves, but assembly accuracy deteriorates
Solution Approach 1:
The patent employs self-service mechanisms where the homology sequences inherently guide the correct assembly through their complementary base pairing properties. The system uses itself (the homology regions) to ensure accurate matching and joining, eliminating the need for complex external verification steps while maintaining high assembly accuracy.
Solution Approach 2:
The patent optimizes specific parameters including homology sequence length (20-100 base pairs), temperature conditions, and enzyme concentrations to achieve the optimal balance between assembly efficiency and accuracy. By carefully controlling these parameters, the method achieves both high productivity and high correct assembly percentages.
3Manufacturing precision
If homology sequences are extended to improve assembly accuracy, then manufacturing precision improves, but process complexity increases
Solution Approach 1:
The patent designs universal homology sequences that can be used across different fragment assemblies and vector types. These standardized homology regions serve multiple functions including recognition, binding, and directional orientation, reducing the need for custom-designed sequences for each application while maintaining high assembly accuracy.
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 efficient and accurate assembly of nucleic acids with high percentages of correct assembly, improving scalability and reducing errors in nucleic acid synthesis.
Implementation Method 1
the bacterial lysate comprises a nuclease
Implementation Method 2
the bacterial lysate comprises a recombinase
Data Source
AI summary
Provided herein are methods, systems, and compositions for seamless nucleic acid assembly. Such methods, systems, and compositions for seamless nucleic acid assembly include those for in vitro recombination cloning, single-stranded hierarchal DNA assembly, or overlap extension PCR without primer removal.