Targeted Dna Integration
Targeted Dna Integration - Related Videos
09:31
Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites
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Cited by 56 •
2016
We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.
07:58
Cell-Free DNA Integrity Analysis in Urine Samples
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Cited by 16 •
2017
A method for analyzing DNA integrity in the cell-free supernatant fraction of urine samples is proposed. The method is suitable for early detection of urological malignancies and has proven accurate for the early diagnosis of bladder cancer.
08:38
Targeted DNA Methylation Analysis by Next-generation Sequencing
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Cited by 83 •
2015
Bisulfite amplicon sequencing (BSAS) is a method for quantifying cytosine methylation in targeted genomic regions of interest. This method uses bisulfite conversion paired with PCR amplification of target regions prior to next-generation sequencing to produce absolute quantitation of DNA methylation at a base-specific level.
07:05
Chromatin Immunoprecipitation to Identify Target Protein Binding Sites on Genomic DNA
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2025
In this video, we demonstrate the chromatin immunoprecipitation technique to identify protein binding sites on specific regions of the genomic DNA of oligodendrocyte precursor cells via the selective immunoprecipitation of protein-bound chromatin fragments. This method helps to study the interaction of several regulatory proteins, including transcription factors involved in gene regulation.
06:10
Non-Viral Engineering of Primary Human T Cells via Homology-Mediated End-Joining Targeted Integration of Large DNA Templates
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2025
A detailed protocol is provided for using CRISPR/Cas9 technology to achieve highly efficient targeted knock-in of large, multicistronic constructs in primary human T cells via the homology-mediated end joining (HMEJ) DNA repair pathway. T cells engineered with this cGMP-adaptable protocol maintain excellent cell expansion, cytotoxicity, and cytokine production.
FAQs
Homology-directed repair uses a donor DNA template to guide insertion of the desired sequence at the genomic site opened by a programmable nuclease. The template supplies sequence information that corresponds to the targeted region, helping align the new DNA with the intended location. This mechanism supports precise gene replacement, tagging, or other controlled genome modifications.
The programmable nuclease identifies the selected genomic location and creates a break there, while the donor DNA template carries the sequence intended for insertion. Their coordinated roles connect site recognition with sequence replacement or addition. This division of function allows researchers to specify both where the modification occurs and what genetic material is introduced.
Both approaches can place DNA at a defined genomic location, but they use different mechanisms. CRISPR-Cas-based strategies typically create a targeted break and use a donor template through homology-directed repair. Site-specific recombinases provide an alternative mechanism for defined insertion. The choice therefore depends on the integration strategy being used in the biological system.
A typical workflow selects the genomic location, designs a programmable nuclease for that site, and prepares a donor DNA template containing the desired sequence. The nuclease creates the targeted break, after which the donor supports insertion through homology-directed repair. Researchers can then use the modified cells for gene replacement, tagging, or regulation studies.
Researchers may choose this approach when a defined genomic modification is important for interpreting cellular behavior. It supports disease modeling, functional genomics, cell-line development, and engineered cell therapies. By placing a sequence at a selected location, the method can help connect a planned genetic change with observed biological outcomes more directly than random insertion approaches.
The approach can test gene function through replacement, attach a tag to study a gene or its product, or modify a locus for regulation studies. These uses make it relevant to functional genomics and disease modeling, where researchers need to examine how a specific genomic change influences cells. It also supports development of engineered cell lines and therapies.