In cells and in the lab, enzymes called ligases are used to join fragments of DNA together. Only DNA fragments that have matching, complementary ends can be joined by ligation.
How do ligases work?
Ligases join fragments of DNA together by catalysing the formation of bonds between neighbouring nucleotides.
Ligation in cells
Cells naturally carry out ligation during DNA replication, when the Okazaki fragments are joined together. Cells also use ligation to repair DNA that has been damaged, either by normal cell metabolism or by environmental factors, such as UV light or radiation. Up to 1 million breaks can occur in the DNA of a single human cell each day.
Ligation in the lab
For DNA ligation to occur in the lab, the reaction mixture must contain: complementary DNA fragments, a ligasebuffer, and a source of energy. The energy for this reaction normally comes from a chemical called ATP (adenosine triphosphate).
Getting the DNA fragments
DNA is prepared for ligation by being cut into fragments with restriction enzymes. Each restriction enzyme cuts DNA at a specific site and makes fragments that have either ‘ blunt’ or ‘ sticky’ ends.
Joining DNA fragments
Ligases can join any DNA fragments with ‘blunt’ ends. They can also join DNA fragments with ‘sticky’ ends, but only if the nucleotides on the strands are complementary. To get complementary ‘sticky ends’ the DNA fragments to be joined must be cut with the same restriction enzyme.
When is ligation used in the laboratory?
Ligation can join together fragments of DNA from different sources. Ligation is often used for DNA cloning.
Where do ligases come from?
Ligases are found in all organisms, but the ligases used in the lab were first isolated from bacteria.