Direct Cloning Using RecE Exonuclease and RecT Annealing Protein
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Solution Overview
Problem
Current methods for direct cloning of DNA regions from genomic DNA preparations are inefficient, particularly for larger DNA regions, and are genetically unstable, limiting their application in complex DNA engineering tasks such as assembling multiple DNA pieces into precise products.
Innovation Solution
The use of full-length RecE, a 5' to 3' exonuclease with an extended N-terminal sequence, in conjunction with an annealing protein like RecT, to enhance homologous recombination efficiency by bringing nucleic acid molecules into contact in the presence of single-stranded oligonucleotides and using rare-cutting sequence specific DNA cleaving enzymes to generate linear nucleic acid molecules for improved homologous recombination.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional cloning methods are used to clone DNA from genomic DNA preparations, then DNA can be amplified and propagated, but the process is laborious and requires multiple screening and subcloning steps
Solution Approach 1:
The invention extracts and utilizes specific proteins (exonucleases and annealing proteins) from their natural context to perform homologous recombination in vitro, separating the desired recombination function from the complex cellular machinery and enabling direct cloning without traditional library screening
Solution Approach 2:
The invention introduces homologous recombination as an intermediary mechanism between DNA fragmentation and cloning vector integration, using controlled in vitro recombination to directly join DNA fragments to vectors without requiring complex screening processes
2Ease of manufacture
If yeast-based direct cloning is used, then DNA can be cloned directly from genomic preparations, but the method is technically challenging and genetically unstable
Solution Approach 1:
The invention copies the essential functions of yeast-based homologous recombination into a controlled in vitro system using purified proteins, replicating the desired cloning capability while eliminating the genetic instability and technical challenges of living yeast systems
Solution Approach 2:
The invention replaces the biological yeast cell system with an in vitro biochemical system using purified exonucleases and annealing proteins, substituting a controllable biochemical mechanism for a living cellular system to achieve stability and ease of use
3Length of stationary object
If current direct cloning methods are used for larger DNA regions, then cloning can be attempted, but efficiency significantly decreases
Solution Approach 1:
The invention segments the DNA manipulation process into controlled in vitro steps (exonuclease treatment, annealing, ligation) that can be optimized independently, allowing efficient handling of large DNA regions without the efficiency loss seen in conventional methods
Solution Approach 2:
The invention changes the parameters of the cloning process by using in vitro homologous recombination with controlled protein concentrations and reaction conditions, enabling efficient cloning of large DNA regions that are inefficient with conventional methods
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 significantly increases the efficiency of linear to linear homologous recombination, allowing for the direct cloning of larger DNA regions and improving complex DNA engineering tasks by enhancing the precision and efficiency of DNA assembly.
Implementation Method 1
a 5′ to 3′ exonuclease with an extended N-terminal sequence
Implementation Method 2
enhance homologous recombination efficiency by bringing nucleic acid molecules into contact
Data Source
AI summary
A method for performing homologous recombination between at least a first nucleic acid molecule and a second nucleic acid molecule which share at least one region of sequence homology. A method for improving the efficiency of homologous recombination.