Ligation Independent Cloning
Ligation Independent Cloning - Related Videos
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Molecular Cloning
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2023
Molecular cloning is a set of methods, which are used to insert recombinant DNA into a vector - a carrier of DNA molecules that will replicate recombinant DNA fragments in host organisms. The DNA fragment, which may be a gene, can be isolated from a prokaryotic or eukaryotic specimen. Following isolation of the fragment of interest, or insert, both the vector and insert must be cut with restriction enzymes and purified. The purified pieces are joined together though a technique called...
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S. cerevisiae are unicellular eukaryotes that are a commonly-used model organism in biological research. In the course of their work, yeast researchers rely upon the fundamental technique of transformation (the uptake of foreign DNA by the cell) to control gene expression, induce genetic deletions, express recombinant proteins, and label subcellular structures. This video provides an overview of how and why yeast transformation is carried out in the lab. The important features of yeast...
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DNA Ligation Reactions
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2023
In molecular biology, ligation refers to the joining of two DNA fragments through the formation of a phosphodiester bond. An enzyme known as a ligase catalyzes the ligation reaction. In the cell, ligases repair single and double strand breaks that occur during DNA replication. In the laboratory, DNA ligase is used during molecular cloning to join DNA fragments of inserts with vectors – carrier DNA molecules that will replicate target fragments in host organisms. This video provides an...
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Culturing and Harvesting of HSPC Clones: A Method to Obtain the Singly-Sorted HSPC Clones
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Rescue and Characterization of Recombinant Virus from a New World Zika Virus Infectious Clone
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This protocol describes the recovery of infectious Zika virus from a two-plasmid infectious cDNA clone.
FAQs
T4 DNA polymerase uses its 3′ exonuclease activity to process the ends of the insert and vector, producing complementary single-stranded overhangs. These exposed sequences provide the base-pairing regions that bring the two DNA molecules together. The enzyme therefore replaces a ligase-dependent joining step with an end-preparation and annealing mechanism while preserving sequence specificity during assembly.
Complementary overhangs give the insert and vector matching sequences that anneal selectively rather than associating randomly. Their sequence-specific pairing supports directional cloning, so the inserted fragment is assembled in a defined relationship to the vector. This matters when a study requires a recombinant construct whose orientation supports subsequent repair, propagation, and analysis in host cells.
Unlike a conventional ligation-based approach, LIC does not require DNA ligase to join the prepared DNA ends. Instead, T4 DNA polymerase generates compatible single-stranded regions, and the insert-vector pair is held together by base pairing before host-cell repair. This distinction simplifies the joining mechanism and is central to LIC’s streamlined construction of recombinant DNA.
After complementary insert and vector ends anneal, the recombinant molecule is introduced into host cells. Those cells repair the annealed DNA and support its propagation, allowing the assembled construct to be maintained for later analysis. Host-cell processing therefore completes the transition from a paired DNA intermediate to a recombinant molecule that can be propagated for study.
A basic workflow begins with an insert and vector, uses T4 DNA polymerase to generate complementary single-stranded overhangs, and allows the ends to anneal through sequence-specific base pairing. The resulting recombinant DNA is then introduced into host cells, where repair and propagation take place. These stages connect molecular assembly with recovery of the construct for study.
LIC can be used to assemble and analyze genes obtained from microorganisms or metagenomic samples. In this context, the method provides a route for placing environmental genetic material into recombinant constructs that can be repaired and propagated in host cells. Researchers can therefore examine genes associated with environmental organisms, including material recovered from communities rather than a single characterized microbe.
Environmental applications include investigating pollutant degradation, microbial adaptation, and engineered pathways. By supporting directional construction and analysis of relevant genes, LIC can help connect DNA sequences with environmental functions or biotechnology goals. The approach is therefore useful both for examining how microorganisms respond to environmental conditions and for studying genetic designs intended to support environmental biotechnology.