Ordered DNA Fragment Assembly with Computer-Designed Overhangs
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Solution Overview
Problem
Existing DNA assembly methods, such as Golden Gate assembly, are limited by the number of fragments that can be assembled in a single reaction, often restricted to 6-8 fragments due to the constraints of standardized overhangs and enzyme biases, leading to decreased yield and increased experimental complexity.
Innovation Solution
A method and system for determining optimized overhang sequences and experimental conditions using a computer-aided approach to assemble a target polynucleotide from a set of double-stranded polynucleotide fragments, allowing for the assembly of up to 100 fragments with high fidelity by using Type IIS restriction endonucleases and T4 DNA ligase, and enabling multiplex amplification and ligation in a single reaction.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If standardized overhangs and traditional Golden Gate assembly methods are used, then the assembly process is simple and well-established, but the number of fragments that can be assembled in a single reaction is limited to 6-8 fragments
Solution Approach 1:
The patent changes the parameters of overhang design by moving from standardized, limited overhang sets to customized overhang sequences optimized for each fragment combination. This allows scaling from 6-8 fragments to 100+ fragments by tailoring overhang properties (sequence, length, composition) to maximize ligation efficiency for each specific assembly scenario
Solution Approach 2:
The patent applies computer-aided design to pre-determine optimal overhang sequences before assembly. By calculating and selecting the best overhang combinations in silico based on ligation fidelity data, the system prepares the optimal configuration in advance, enabling high-fidelity assembly of large numbers of fragments without increasing experimental complexity
2Productivity
If the number of fragments in an assembly reaction is increased, then the productivity and efficiency of DNA molecule construction is improved, but the experimental complexity and difficulty of identifying suitable overhangs increases
Solution Approach 1:
The patent replaces manual, trial-and-error overhang selection with a computer-aided design system. The software automatically calculates optimal overhang sequences by considering ligation fidelity data, fragment compatibility, and assembly conditions, substituting computational algorithms for manual experimental optimization
Solution Approach 2:
The patent introduces a computer-aided design system as an intermediary between the researcher and the assembly reaction. This intermediary software processes fragment sequences, evaluates overhang options using ligation fidelity databases, and outputs optimized overhang designs, simplifying the workflow for large-scale assemblies
3Reliability
If T4 DNA ligase is used for joining DNA fragments, then the ligation process is well-established and reliable, but the enzyme exhibits bias with respect to ligating various complementary overhangs with varying efficiency
Solution Approach 1:
The patent addresses T4 ligase bias by changing the overhang parameters (sequence composition, length, GC content) to match the enzyme's preferences. The computer-aided design system selects overhangs with sequences that T4 ligase ligates with high fidelity, compensating for enzyme bias through rational overhang design rather than enzyme substitution
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
Enables the efficient and accurate assembly of large DNA molecules from multiple fragments in a single reaction, reducing experimental complexity and enhancing yield by optimizing overhang sequences and conditions for high-fidelity ligation.
Implementation Method 1
fragments of DNA are created using restriction endonucleases that generate single strand overhangs on double stranded DNA
Implementation Method 2
Ligation then occurs between overhangs on different fragments to assemble a single double stranded molecule from the fragments
Implementation Method 3
Ligation then occurs between overhangs on different fragments
Data Source
AI summary
A composition and its uses and additionally a kit are provided. The composition is a synthetic self-complementary oligonucleotide that has a double-stranded region and a loop, wherein the double-stranded region contains a binding sequence for PaqCI. Additionally, the oligonucleotide includes unligatable 3′ and 5′ ends that cannot be cleaved by PaqCI. This oligonucleotide composition has been combined with PaqCI or a variant of PaqCI Type IIS restriction endonuclease in a reaction mixture, where the reaction mixture includes PaqCI or variant that can further be combined with a ligase and optionally a deadenylase, crowding molecule such as PEG and/or a repair enzyme such as Endo MS. The kit includes the oligonucleotide and Type IIS restriction endonuclease in the same or different containers.