Molecular biology · BEGINNER LESSON
Ligation
Ligation is the enzyme-catalyzed joining of two biological molecules, most clearly DNA fragments, by forming a covalent phosphodiester bond between a 5′ phosphate and a 3′ hydroxyl end. In DNA work, success is judged by whether the ends become one continuous joined molecule.
What ligation is
Ligation is a joining reaction carried out by ligases, a class of enzymes. In DNA, ligases connect one DNA piece to another by making phosphodiester bonds, the chemical links that hold nucleotides together in a strand. The reaction can also join the two ends of one molecule. For DNA cloning, the main thing being measured is whether separate DNA fragments become a continuous molecule after mixing with ligase and the needed energy source.
[1] DNA Cloning [2] DNA Ligases: Progress and Prospects - PMC - NIH
How the joining works
DNA ligation happens only at ends that can be paired or positioned close enough for the enzyme to act. If restriction enzymes have made sticky ends, short single-stranded overhangs can base-pair briefly, which helps ligase seal the gap. With blunt ends, there is no temporary base-pairing, so ligation is less efficient. DNA ligases use energy from ATP, or from NAD+ in some ligases, to carry out the chemical steps that seal a nick between a 5′ phosphate and a 3′ OH group.
[1] DNA Cloning [2] DNA Ligases: Progress and Prospects - PMC - NIH
What controls the reaction
The ends must have the right chemical groups: two ends with 5′ phosphates can be joined, and a phosphatased end can join a non-phosphatased end, but neither end can be missing a 5′ phosphate. In the DNA-cloning setting, ligase does not actively bring fragments together; random molecular motion must place the ends close enough first. For proteins, related ligation ideas are used in expressed protein ligation, where a protein fragment and a peptide are linked by chemoselective ligation.
[1] DNA Cloning [3] Methods and Applications of Expressed Protein Ligation - PMC
Limitations and scope
Ligation is powerful but not automatic. Sticky ends usually ligate more easily than blunt ends, and the required energy chemistry differs among ligases. The broader term also appears in protein semisynthesis, where ligation can join protein fragments, but those methods use different chemistries and specialized enzymes. So ligation is a general joining strategy, not one single reaction for every biomolecule.
[1] DNA Cloning [2] DNA Ligases: Progress and Prospects - PMC - NIH [3] Methods and Applications of Expressed Protein Ligation - PMC
SEE THE IDEA
How DNA ligation connects fragments
Restriction enzymes create DNA fragments with blunt ends or sticky ends.
Random diffusion brings compatible ends near each other.
DNA ligase uses ATP or NAD+ to seal the chemical gap.
A continuous DNA strand is formed with phosphodiester bonds.
Each step depends on the previous one: cut ends must exist, then meet, then be sealed by ligase.
Ligation is a chemical sealing step that can join DNA fragments when their ends and energy source are suitable.
TRY IT YOURSELF
Test your understanding
Choose an answer, then check the explanation.
PUT IT TOGETHER
Match each concept to its meaning
Drag a label onto its description, or select a label and then select a description. This also works with a keyboard.
4 pairs to match
Sources and further reading
This general lesson was generated from public source material. It explains a concept; it does not interpret a private paper or provide medical advice.