Can DNA ligase ligate blunt ends?
Blunt-ended fragments can be joined to each other by DNA ligase. However, blunt-ended fragments are harder to ligate together (the ligation reaction is less efficient and more likely to fail) because there are no single-stranded overhangs to hold the DNA molecules in position.
How are blunt ends produced?
You can create blunt ends by filling in single stranded overhangs remaining after physically shearing (see Fig. 2) or cutting with restriction endonucleases that generate sticky ends. The single-stranded overhangs can be repaired using a mixture of DNA polymerases such as T4 polymerase and the Klenow fragment.
What is ligation and types of ligation?
DNA ligation is the act of joining together DNA strands with covalent bonds with the aim of making new viable DNA or plasmids. There are currently three methods for joining DNA fragments in vitro. The first of these is DNA ligase that covalently joins the annealed cohesive ends produced by certain restriction enzymes.
What is the goal of the ligation reaction?
This reaction, called ligation, is performed by the T4 DNA ligase enzyme. The DNA ligase catalyzes the formation of covalent phosphodiester linkages, which permanently join the nucleotides together.
What is the difference between cohesive-end and blunt-end ligations?
In blunt-end ligations, the association of 5’ phosphate groups and 3’ hydroxyl groups is more transient than in cohesive-end ligations. Because they lack the hydrogen bond stabilization of cohesive ends, blunt-end ligations are more sensitive to reaction conditions, especially to the concentrations of the reaction components.
What is the blunt end of a PCR fragment?
On a related note, PCR-generated DNA fragments are always blunt ended, and may be used directly in blunt-end ligations – unless you use Taq polymerase. Taq adds an extra adenine to the 3’ end of the PCR product, so you’ll need to at a bit of 3′-5′ exonuclease activity (e.g. from a spot of Pfu) to blunt the ends.
What is the function of DNA ligase and blunt end?
Both are useful in molecular genetics for making recombinant DNA and proteins. Blunt ends are generated by cutting both DNA strands in the middle of the recognition sequence. DNA ligase helps to join together the complementary ends of insert DNA and plasmid DNA.
Is it possible to perform DNA ligations with large DNA fragments?
Performing these ligations is notoriously difficult, particularly with large DNA fragments. But it is possible. And in this article I’ll give you some tips that I hope will increase your chances of success.