Oligonucleotide Recombineering
Oligonucleotide Recombineering - Related Videos
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Chemical Triphosphorylation of Oligonucleotides
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Oligonucleotide 5′-triphosphates are ubiquitous components in essential biological pathways and have seen increasing use in biotechnology applications. Here, we describe techniques for the routine synthesis and purification of oligonucleotide 5′-triphosphates, starting from oligonucleotides prepared by standard automated synthesis techniques.
08:56
Recombineering and Gene Targeting
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One of the most widely used tools in modern biology is molecular cloning with restriction enzymes, which create compatible ends between DNA fragments that allow them to be joined together. However, this technique has certain restrictions that limit its applicability for large or complex DNA construct generation. A newer technique that addresses some of these shortcomings is recombineering, which modifies DNA using homologous recombination (HR), the exchange between different DNA molecules based...
14:23
Recombineering Homologous Recombination Constructs in Drosophila
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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.
05:33
Using Modified Synthetic Oligonucleotides to Assay Nucleic Acid-Metabolizing Enzymes
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Here, a protocol for assaying nucleic acid metabolizing enzymes is presented, using examples of ligase, nuclease, and polymerase enzymes. The assay utilizes fluorescently labeled and unlabeled oligonucleotides that can be combined to form duplexes mimicking RNA and/or DNA damages or pathway intermediates, allowing for the characterization of enzyme behavior.
11:33
Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.
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Microinjection is a well-established and effective method for introducing foreign substances into fertilized zebrafish embryos. Here, we demonstrate a robust microinjection technique for performing mRNA overexpression, and morpholino oligonucleotide gene knockdown studies in zebrafish.
FAQs
The key mechanism is timing: the oligonucleotide must pair with a complementary sequence in newly replicated DNA or at a replication fork. DNA synthesis can then copy the designed sequence into the genetic material. Repair and subsequent replication resolve the strategically placed mismatches, allowing a temporary pairing event to become a stable genotype.
Recombination functions provide the cellular context needed for the designed oligonucleotide to anneal to its target during replication. Without that context, the intended pairing and incorporation step may not occur efficiently. In practice, cells expressing these functions enable targeted changes in either chromosomal DNA or plasmid DNA, depending on the experimental design.
The mismatch pattern carries the information for the desired allele. Depending on how the oligonucleotide differs from the original template, repair and later replication can produce a base substitution, an insertion, or a deletion. This design principle lets researchers connect a specific sequence alteration with its resulting genetic or phenotypic effect.
A major distinction is that this approach can introduce targeted sequence changes without intentionally creating a double-strand break. The oligonucleotide instead participates in DNA synthesis at a replication-related intermediate. This provides an alternative route for allele construction when researchers want precise sequence modification while avoiding reliance on a chromosome-cleaving event.
A typical workflow begins by selecting the chromosome or plasmid sequence to change and designing a short oligonucleotide with the intended sequence difference. Cells expressing recombination functions then provide the setting for annealing during replication. DNA synthesis, mismatch repair, and subsequent replication convert the design into the requested substitution, insertion, or deletion.
The method is useful when researchers need to build defined alleles quickly, test how mutations affect function, or generate many sequence variants. Its flexibility supports multiplexed editing and variant-library construction, while its precision helps relate genotype to phenotype. These capabilities make it valuable for microbial genome engineering and systematic functional analysis.