Dna Ligation
Dna Ligation - Related Videos
07:34
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...
13:10
Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay
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Cited by 3 •
2017
Here, it is demonstrated how the in situ Proximity Ligation Assay (PLA) can be used to detect and visualize the direct protein-protein interactions between ATM and p53 in suspension cell cultures exposed to genotoxic stress.
11:53
Cecal Ligation Puncture Procedure
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Cited by 200 •
2011
The mouse model of cecal ligation and puncture as a valuable tool for the study of human sepsis.
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.
04:49
Proximity Ligation Assay to Study In Situ Protein-Protein Interactions
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2025
This video describes the proximity ligation assay to study in situ protein-protein interactions in nematode cells. The close proximity of the proteins of interest generates the circular DNA template with the help of a PLA probe attached to a secondary antibody. The probe is further amplified using the amplification solution, which produces a long single-stranded DNA followed by the hybridization of the detector probe, enabling the localization of protein-protein interactions in cells.
FAQs
The ligase reaction requires these two chemically distinct groups to meet at a break in the DNA backbone. Their alignment allows the enzyme to create a covalent phosphodiester linkage, converting a discontinuity into a continuous strand. This requirement explains why the chemical ends present on DNA fragments determine whether joining can occur.
DNA ligases require an energy input to form the new backbone linkage. Depending on the ligase, that energy comes from ATP or NAD+. The relevant cofactor is therefore a defining property of the enzyme system and must match the ligase being used, particularly when selecting conditions for laboratory assembly or interpreting cellular ligation processes.
Cohesive ends contain compatible single-stranded regions that can pair with one another before ligation, whereas blunt ends lack such overhanging complementarity. Both can be joined under suitable conditions, but the end structure affects how DNA fragments align for sealing. This distinction is important when planning recombinant DNA assembly and evaluating possible fragment connections.
Within cells, ligation acts after DNA synthesis or repair has produced adjacent DNA segments that still contain a backbone break. Sealing those interruptions completes the continuity of the molecule and helps preserve chromosome integrity. Its involvement in both replication and repair shows that ligation supports normal genome maintenance, not only engineered DNA construction.
A DNA fragment is joined to a plasmid vector so that the resulting recombinant molecule can be used in downstream molecular biology studies. The ligation step connects the selected DNA pieces through their compatible or blunt ends under suitable conditions. This assembled DNA supports experimental work involving cloning, sequencing, or gene expression.
Ligation-based assembly provides a way to create recombinant DNA molecules for several distinct purposes. Researchers can use the assembled constructs in cloning studies, prepare DNA arrangements relevant to sequencing, or examine gene expression from an inserted fragment. These applications make ligation a practical bridge between fragment joining and broader experimental analysis in biology.