Ligation Amplification
Ligation Amplification - Related Videos
09:44
Rapid Amplification of cDNA Ends
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2023
Source: Pablo Sanchez Bosch2, Sean Corcoran2 and Katja Brückner1,2,3 1Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research 2Department of Cell and Tissue Biology, 3Cardiovascular Research Institute, University of California San Francisco, San Francisco, CA, USA Rapid Amplification of cDNA Ends (RACE) is a technique that allows amplification of full-length cDNA from mRNA by extending to the 3’ or 5’ end, even without prior knowledge of the sequence (Frohman et al.,...
02:41
Amplification of a Suicide Plasmid in a Bacterial Host
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2025
Source: Tomás, J. M., et al. Generation of Null Mutants to Elucidate the Role of Bacterial Glycosyltransferases in Bacterial Motility. J. Vis. Exp. (2022).This video demonstrates the initiator-dependent amplification of a suicide plasmid in E. coli. The plasmid, which carries an antibiotic resistance gene, replicates only in the presence of an initiator protein encoded by the host. After electroporation and incubation in recovery medium, the mixture is plated on antibiotic-containing agar.
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...
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.
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.
FAQs
Specificity comes from the ligation junction rather than hybridization alone. Two oligonucleotide probes must bind adjacent positions on a complementary template, placing their ends in the correct geometry. DNA ligase then catalyzes formation of the phosphodiester bond. If alignment is incorrect, joining is not efficiently supported, so the reaction preferentially produces products matching the intended sequence.
At a potential point-mutation site, the probe boundary can be arranged so that correct base pairing is required for ligation. A matching target supports precise end alignment, whereas a mismatched target is less likely to satisfy the ligase’s joining requirement. This makes the method useful for distinguishing selected sequence variants, although it supports detection of some, rather than all, point mutations.
Thermal cycling drives the repeated reaction in complementary phases. Heating separates previously formed products from template-associated structures, while subsequent reaction conditions enable the probes to hybridize again at neighboring positions. Ligase can then join correctly aligned ends in another round. Repetition increases the amount of target-associated ligated material rather than relying on a single joining event.
A basic workflow begins with a complementary nucleic acid template and two oligonucleotide probes designed to bind next to one another. The probes hybridize, and DNA ligase joins their correctly aligned ends. Thermal cycling then separates products and permits additional hybridization and ligation rounds. The resulting increase in joined products provides amplified material for sequence-specific analysis.
Researchers may choose this technique when sequence discrimination is important, particularly for molecular diagnostics, genotyping, or analysis of low-abundance nucleic acids. Its dependence on correct base pairing at the joining site links product formation to a specific target sequence. Consequently, the method can support detection of selected sequence differences while amplifying material that may initially be scarce.
In molecular diagnostics and genotyping, the method connects a biochemical joining event with the identity of a nucleic acid sequence. Correctly adjacent probes can be ligated only when their ends align with the complementary template, allowing selected target sequences or some point mutations to be distinguished. Repeated cycles increase the available ligated material for subsequent analysis of those targets.