T4 DNA ligase is an enzyme that fixes broken DNA and seals it – similar to super glue. This particular DNA ligase was isolated from bacteriophage T4. During DNA replication or recombination, a break or a ‘nick’ in the backbone of DNA frequently occurs. Afterwards, the DNA ligase comes in and plays an important role on repairing these nicked DNA strands by joining both ends of the DNA.
In this article
What DNA ligase does: Understanding its purpose
DNA ligase repairs broken DNA by forming a phosphodiester bond between a nearby 5’ phosphate and 3’ OH of the nicked or cut DNA strand. In addition to duplex DNA, T4 DNA ligase can also seal single stranded cut in RNA or DNA/RNA hybrids.
In molecular biology labs, this enzyme is mostly used for cloning to ligate either cohesive or blunt ends of DNA inserts into a vector. Cohesive ends means the end parts of DNA contain short single-stranded tails or overhangs (sticky ends), whereas blunt ends do not have overhangs.
Using T4 DNA ligase to insert DNA into a vector. In this illustration, PCR is used to amplify DNA inserts. Then, the PCR products can be ligated into a vector by using T4 DNA ligase.
How does DNA ligase work?
T4 DNA ligase works in these sequential steps:
- Enzyme adenylation: This first step involves an addition of an adenosine monophosphate (AMP) molecule from a high-energy co factor, such as ATP or NAD+, into the ligase.
- The transfer of AMP: In the second step, the AMP molecule is then transferred to the 5’ phosphate at the site of a nick.
- Nucleophilic attack of the 5’ terminus: In the last step, due to the presence of AMP on the 5’ phosphate, 3’ OH performs a nucleophilic attack on 5’ phosphate to form a phosphodiester bond.
DNA Ligation by T4 DNA Ligase. The three steps in this process are the enzyme adenylation, the transfer of AMP, and nucleophilic attack of the 5' terminus.
How to set up a ligation reaction using T4 DNA ligase
Below is a common procedure to set up a ligation reaction:
- Into a reaction tube, combine the following: vector, insert DNA, T4 Ligase Buffer, T4 DNA Ligase, and nuclease-free water.
- Gently mix the reaction and centrifuge briefly.
- Incubate the reactions.
- Heat-inactivate the ligation reactions.
Why is heat inactivating T4 DNA ligase necessary?
The heat inactivation step of T4 DNA ligase is necessary to end ligating activity, particularly if the use of ligase can inhibit downstream chemical reactions. In electroporation, heat inactivating the reactions help increase the transformation efficiency. In chemical transformation, you can use the ligation reaction immediately without heat inactivation step. But, if you want to store the ligation reaction for and extended duration, you will want to heat inactivate your reaction.
At what temperature is T4 DNA ligase most active?
Ligation reactions are optimal at 12 to 16 °C, in order to maintain the balance between the activity of T4 DNA ligase and the stability of annealed DNA strands (Lund et al., 1996).
Performing ligations at high temperatures may increase the enzyme activity. But it reduces the ligation efficiency overall, because the DNA molecules move rapidly and it becomes difficult for the end parts of DNA fragments to form a covalent bond with each other and stay connected.
Alternatively, using lower temperatures may slow down the DNA molecules to better form covalent bonds, but also decrease the ligase activity. Therefore, it’s best to check the manufacturer’s instruction for the recommended temperature before performing a ligation.
For optimal use of GoldBio T4 DNA ligase, set up the ligation reaction at 16 °C. The incubation time can be as quick as 10 minutes for ligating cohesive ends, or at least 2 hours for ligating the blunt ends. However, for both types of DNA ends, using longer incubation times of 15-18 hours at 16 °C, will significantly improves the ligation efficiency.
Why do ligation reactions using T4 DNA ligase fail?
Ligation reactions fail due to these following possible reasons:
1.The presence of ligase inhibitors in the reactions
To prevent contaminations of ligase inhibitors in the reactions, such as high salts or EDTA, make sure you purify your DNA insert and plasmid. One way to remove contaminants is by performing ethanol precipitation.
So, what are ligase inhibitors? Ligase inhibitors are substances in a solution, such as high salts or EDTA, which can inhibit the activity of ligase.
2.The DNA concentration is too low or too high
If the concentration of DNA of the ligation reaction is too low, there might not be enough DNA ends to anneal to the vector. Whereas, if the concentration of DNA is too high, the ligation reaction can produce unwanted long and linear DNA molecules. Therefore, try different ratios of vector and insert DNA to find the ratio that works best. To start with, you can test a 1:1 and 1:3 molar ratio of vector:insert DNA.
To find out how much insert DNA to add in a ligation reaction if you want the ratio to be 1:3, use the formula below:
3.ATP concentration in the buffer is too low
If the ATP concentration in the ligation reaction is too low, the ligation will fail. Therefore, make sure to use the compatible buffer that is supplied with your T4 DNA ligase. Additionally, double check the expiration date of the T4 DNA ligase to confirm whether the buffer or ligase should be discarded. Lastly, frequent freezing and thawing the ligation buffer might decrease the activity of the ATP in the buffer. Adding a bolus of fresh ATP to your ligation reaction may be necessary.
4.The inactive enzyme
The ligation reaction might also fail due to the inactive enzyme. Make sure to test your ligase for functionality before use.
How to test if your T4 DNA Ligase still works
One way to test if your T4 DNA ligase still works is to test your ligase by using phage Lambda DNA digested with a HindIII restriction enzyme.
Below are the steps to test your ligase:
- Combine 2 µl of buffer, 1 µl Lambda-HindIII digested DNA, 1 unit of the T4 DNA ligase and water for a 20 µl ligation reaction.
- Set up also a negative control with no addition of ligase.
- Incubate the reactions at 22 °C for 10 minutes.
- After incubation, use 10 µl of the reactions to perform gel electrophoresis.
- If your ligase is still active, you should observe a distinct band with a higher molecular weight for the reaction with the ligase compared to the one without the ligase.
How to store T4 DNA ligase and the buffer
Store T4 DNA ligase and the buffer in -20 °C freezer. This enzyme should be added last in the ligation reaction. Therefore, keep the enzyme in the freezer until it’s time to add it in the reaction, and keep the tube on ice. Return the enzyme to the freezer as soon as possible after use.
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Ymer, S. (1991). Heat inactivation of DNA ligase prior to electroporation increases transformation efficiency. Nucleic Acids Research, 19(24), 6960. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC32934...