Donor Plasmid Homology Arms
Donor Plasmid Homology Arms - Related Videos
14:23
Recombineering Homologous Recombination Constructs in Drosophila
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Cited by 9 •
2013
Homologous recombination techniques greatly advance Drosophila genetics by enabling the creation of molecularly precise mutations. The recent adoption of recombineering allows one to manipulate large pieces of DNA and transform them into Drosophila6. The methods presented here combine these techniques to rapidly generate large homologous recombination vectors.
02:31
Homologous Recombination
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2020
The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...
05:12
All-in-One CRISPR Genome Editing: A Method for Homology Directed Repair-Based Gene Knock-In in Cultured Cells Using CRISPR-Cas9 System
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2025
In this video, we demonstrate all-in-one CRISPR-Cas9 based genome editing in cultured cells where Cas9 and sgRNA are provided as a single plasmid construct to the cells. The CRISPR-Cas9 system and desired gene to be inserted was introduced in cells through electroporation technique to facilitate successful gene editing.
04:14
Generation of Antibiotic-Marked Mutants in Cyanobacteria via Homologous Recombination
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2025
Source: Lea-Smith, D. J., et al. Generation of Marked and Markerless Mutants in Model Cyanobacterial Species. J. Vis. Exp. (2016)The video demonstrates a method for generating antibiotic-marked mutants in cyanobacteria. The protocol begins with culturing cyanobacteria, followed by centrifugation and washing steps that preserve surface pili. A plasmid carrying an antibiotic resistance gene flanked by homologous sequences is then introduced. During incubation, the plasmid is internalized via pili...
08:53
Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1
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Cited by 15 •
2011
We provide a method for testing BRCA1 variants in a tissue culture based assay for homologous recombination repair of DNA damage by depleting endogenous BRCA1 protein from a cell using RNAi and replacing it with a BRCA1 point mutant that contains a coding change.
FAQs
Their sequence accuracy and placement help the repair template correspond to the intended genomic locus rather than an unrelated region. The two flanking matches provide genomic context on either side of the planned change, while the donor’s inserted sequence supplies the modification to be copied. Consequently, poorly designed arms can reduce precise targeting even when a nuclease cut is correctly made.
Homology arm length, sequence accuracy, and position are key design variables because they determine how well the donor plasmid corresponds to the target locus. These variables should be considered together rather than treated as interchangeable features. A design with inaccurate or poorly placed matching regions may lower targeting efficiency, making subsequent screening more important for identifying correctly modified cells or organisms.
The programmable nuclease initiates the repair process by creating a double-strand break at a defined genomic site. That break provides the targeted location where the cell can engage the donor plasmid during repair. Because the cut is site-specific, nuclease targeting works together with accurate homology arms to focus the planned modification on the selected genomic locus.
A typical workflow begins by designing homology arms around the intended genomic change and placing the desired sequence between them in a donor plasmid. A programmable nuclease then creates a site-specific break in the target genome, after which cells can use the donor as a repair template. Researchers finally screen resulting cells or organisms for correct integration.
Donor plasmid homology arms support several precise editing goals, including gene insertion, deletion, tagging, and correction. The same general strategy can therefore be adapted to different planned changes rather than being limited to one edit type. Its use in cultured cells and model organisms also makes it relevant across biological techniques where precise genomic modification is the experimental goal.
Correct integration cannot be assumed simply because a donor plasmid was used or a modification was attempted. Screening must verify that the intended sequence is present at the genomic target and distinguish that outcome from random plasmid insertion. This validation step is essential for interpreting whether the experiment produced the planned genetic change rather than an unrelated integration event.