Restriction Digestion and ligation

Restriction Digestion

You might be tempted to think it’s just a ‘restricted digestion’ of DNA (DNA-what else do you imagine bio experiments to be about!). Well no! The nomenclature has an entirely different significance that we shall soon find out.
We use ‘restriction enzymes’ to cut selective parts of DNA. In our experiment we shall cut a plasmid and make it linear.



Let us know a bit more about these ‘restriction enzymes’. They seem to have been specially invented by our genes to break down harmful phage-DNA. In this way they ‘restricted’ viral infection, hence the name 'restriction enzymes’ (for enzymes which restrict)- and their corresponding enzymatic action, or ‘digestion’, is called ‘restriction digestion’ (digestion to restrict, and not restricted digestion).
Restriction enzymes recognise specific sites on the DNA, where they cut. So, restriction enzymes are highly specific. Some well-known restriction enzymes include EcoRI, RY13 and HInD III.
Restriction enzymes can cut DNA in two ways:

(1)To produce blunt ends
A sharp symmetric cut in the DNA- both the helices of the DNA are cut in the same place.

(2)To produce cohesive/sticky ends
DNA is cut asymmetrically, so the broken ends can still rejoin. There is a tendency to form bonds again at the broken end. This is very useful in cloning, and this is the sort of enzyme we are going to work with.



This is our plasmid, the common PUC18. We observe it has 3 important parts that can be demarcated.



We must make sure that the restriction enzyme we are using does not affect the origin of replication or the antibiotic marker.
The MCS is a speciality. This region contains many ‘cut-able’ sites, each of which can be recognised by some restriction enzyme. Hence, a wide variety of restriction enzymes can be used to cut this DNA at this point.
So we start the experiment. In the given centrifuge tube, we take 10µl of PUC18 plasmid DNA, 2µl of 10X array buffer, 0.8µl of EcoRI (that’s our enzyme) and 7.2µl of water to make it up to 20µl. Mix the stuff by pipetting. Now put it in the incubator at 370C for 1 hour.
That’s it! We are done with the protocol! Cool speed, innit?
We can now do the electrophoresis to check if our plasmids have really been cut.



One important thing to be noted here, before saying goodbye, is the composition of our dye:
1.Xylene Cynol ≈ corresponds to 1-2 Kbp (kilo base pairs) of DNA
2.BPB (bromo phenol blue) ≈500bp
3.Orange G ≈ 50bp
4.Glycerol/sucrose- these heavy molecules help take the contents of our sample to the bottom of the well while loading.

Ligation

How integration follows differentiation in a mathematics course, ligation must follow restriction digestion.
Here, we use another set of enzymes known as DNA ligases- these re-establish the phosphodiester bonds in DNA, thus laterally sealing the broken ends of DNA. The reaction is pH-specific (so we’ll use a buffer in our experiment) and ligases require ATP as a cofactor.
We shall start our experiment without further ado. Let’s take the DNA we cut in the previous experiment and try rejoining to get back our plasmid. In a centrifuge tube, we take 10ml of digested DNA from the previous experiment. Add 2µl of cohesive end buffer (this buffer already contains all the ATP we need), 7.5µl of distilled water and 0.5µl of T4 DNA ligase- this ligase is commonly used by bacteriophages. Incubate it overnight at 40C. That must do it.
Next evening we come back and do the electrophoresis to see how successful our experiment has been.



And it has been quite successful indeed!


A short note on RDT

RDT or Recombinant DNA Technology is a branch of biology that is rapidly growing in its popularity. Here, DNA is cut or modified using restriction enzymes and ligases. This is more popularly known as ‘cloning’ (although the general public image of cloning is quite contrary to this due to the fact that the word ‘clone’ is more often associated with the cloned sheep Dolly, and hence used to mean ‘a process of making copies of living beings’.) Directionality is very important in RDT. Sometimes we use more than one enzyme to make the reaction more specific. We can thus ‘cut and paste’ DNA fragments from one place to another, or insert more DNA into the DNA chain.