DNA Ligation: How it Works

It takes a real effort to keep your basic knowledge of molecular and cell biology fresh, in addition to everything else you have to do. Wouldn’t it be great to if there was a place where you could find easy-to-read articles that allow you to brush-up on those basics in just a couple of minutes?

…I hope you said “yes”, because this is the aim of my “The Basics:” series of articles, which we have already, and will continue to, bring to you periodically. This article explains the basics of DNA ligation.

Your DNA Ligation Buddy: DNA Ligase

DNA ligase (EC is the enzyme at the heart of the DNA ligation reaction. It covalently joins the phosphate backbone of DNA with blunt or compatible cohesive ends (see Figure 1) and it’s natural role is in repairing double strand breaks in DNA molecules. In molecular biology it is commonly used for the insertion of restriction enzyme-generated DNA fragments into vector backbones. Commercial ligases are supplied with a reaction buffer containing ATP and Mg2+, which are both essential for ligase activity. Since ATP can be damaged by repeated freeze-thaw cycles, it is advisable to make aliquots of the buffer (see my article “5 DNA ligation tips“).

dna ligation mechanism

Figure 1. Cohesive and blunt ends, ready for DNA ligation!

The two steps of the DNA ligation reaction

The DNA ligation reaction itself has two basic steps. Firstly the DNA ends have to collide by chance and stay together long enough for the ligase to join them. This is the most inefficient part of the reaction, but is easier at low temperatures. Why? Well, as you will probably know, all molecules move faster at higher temperatures so you can imagine that it is going to be easier for two DNA ends to collide and stay together if they are gently floating through the solution at low temperature, rather than whizzing about as they would be at higher temperatures. For cohesive ends, there is an additional reason; lower temperatures stabilize the hydrogen bonding between the complementary nucleotides, which really helps to keep things in place.

Mechanism of DNA ligation

Figure 2. Enzymatic reaction of DNA ligation

The second step is the enzymatic reaction, which is shown schematically in Figure 2.. DNA ligase catalyzes the joining of the 3′-OH to the 5′-phosphate via a two step mechanism. First, the AMP nucleotide, which is attached to a lysine residue in the enzyme’s active site, is transfered to the 5′-phosphate. Then the AMP-phosphate bond is attacked by the 3′-OH, forming the covalent bond and releasing AMP. To allow the enzyme to carry out further reactions the AMP in the enzyme’s active site must be replenished by ATP.

Here’s why we carrying out DNA Ligation at low temperatures can help

The DNA ligase enzyme has optimal activity at 25°C so the ligation reaction is carried out at a temperature that is a trade-off between the optimal temperatures for bringing the DNA ends together (1°C) and the enzymatic reaction (25°C). Normally 1hr at 16°C is fine but since bringing the DNA ends together is the least efficient part of the reaction favoring this by lowering the temperature to 4°C can give even more efficiency. However, the enzyme will work very slowly at this temperature so a long (e.g. overnight) incubation time is required.

Originally published on 31 October 2007; updated and republished on 5 December 2014.


  1. Chuck Streich on May 9, 2017 at 4:07 pm

    One cool trick is to cycle at 25degC and 16degC. I do 5 min at 25 and 3 min at 16. Repeat this in a thermocycler several dozen times over a couple hours. This allows the ligase to adenylate the DNA at 25 deg and then lower the temp to promote annealing of the sticky ends and final ligation step turnover. Rinse and repeat. After doing this for a while, I later found a published manuscript with quantitative data that supports this as an improved methodology.

    Heres to positive colonies!!

    • Umber on June 6, 2017 at 9:04 am


      I am just wondering how many cycles do you do for this type of ligation? and do you add any SOC (scaffold connector etc)?

    • Dr. David on July 17, 2017 at 10:33 pm

      Can you share us the name of that paper you say it is published the quantitative data? Thanks a lot.

  2. ivi on March 27, 2016 at 5:15 am

    thank you sir!

  3. Archana on February 7, 2016 at 3:59 pm

    great, its good for me ,actually m thankfull of yours for this

  4. Tatiana Claro da Silva on March 3, 2014 at 11:56 am

    Excellent! It is always nice to have a refresher on the basics. Thank you BitesizeBio!

  5. Johnny on July 7, 2013 at 11:21 pm

    Thanks for the detailed explanation. I’m really glad I found your website.

Leave a Comment