DNA ligase only recognizes and acts on the ends of linear DNA molecules, usually result in a complex mixture of DNA molecules with randomly joined ends. The desired products (vector DNA covalently linked to foreign DNA) will be present, but other sequences (e.g. foreign DNA linked to itself, vector DNA linked to itself and higher-order combinations of vector and foreign DNA) are also usually present.
1. Designing a cloning strategy that uses asymmetrical overhangs on either end can limit a lot of undesirable reactions since only one orientation is favorable. Most ligases used in cloning are either bad at, or totally unable to, ligate free ssDNA ends to each other. A lot of cloning methods take advantage of this. Higher order products/random orientations are part of why ligation of blunt ended products is typically avoided when designing a cloning strategy, if possible.
2. Re-ligation of the original vector can be limited by gel extraction (desired fragment is specifically purified) or by phosphatase treatment of the vector but not the desired insert (T4 ligase works by ligating a 5' phosphate to a 3' hydroxyl)
3. A higher insert:vector ratio favors reactions where the new insert is ligated into the vector as opposed to the original insert (which in a restriction digest will be in a 1:1 ratio). 3 insert:1 vector ratios are pretty common in ligations.
- Badges
- Science topic
- Similar topics
- Vectorization