Gene Cloning

The process of cloning a gene involves many biotechnological techniques. Depending on what information is already availible on the gene of interest, various methods can be used to isolate and amplify it.->(click here for animation)

Definition:

To clone a gene means to cut it out of its original place in the host DNA and ligate it into a suitable "vector" where multiple copies of it can be quickly reproduced.

Technique:

1) Firstly we must get the DNA we want in sufficient quantities to clone.

Well, if we had a little bit of the DNA and knew enough of the sequence to make primers we could use PCR to amplify it.

If we started with RNA (eg mRNA) then we would need to use reverse transcriptase to 'reverse transcribe' it into DNA then PCR amplify it.

2) Next we decide on a vector.

A vector is usually a piece of DNA that replicates in a bacterium. Plasmids are usually used, they are small pieces of circular DNA that are often found in bacteria which are self-replicating and are maintained in the cell in a stable and characteristic number of copies. Some bacteria have very high numbers of plasmids which make them ideal cloning vectors.

So, you can see, a gene inserted into a plasmid will be replicated many of times in a single bacteria. And it is possible to grow millions of bacteria in a 1 litre flask. So this is a good way to make lots of copies of a gene of interest.

Routinely in the lab a weakened strain of the bacteria E.coli is used for these experiments. If it is not grown in the lab under special conditions it will quickly die.

3) Application of restriction digestion to cut the DNA.

Next we use restriction enzymes to cut out the gene we want from the amplified product. We use the same enzymes to cut the vector so that the ends will be compatible. This means that if we use a "sticky ends" restriction enzyme, then the DNA sequences of the trailing bits will be compatible and will want to stick to each other. This makes it much easier to do. If we can't find a suitable site for a sticky end restriction enzyme then we can use a "blunt-ended" one, which is a bit harder to make work.

Once the restriction is done the piece of DNA that you want to clone needs to be separated from the parts you do not want this is done using gel electrophoresis as this will make it easier to clone (see screening, below). This is usually done by running the digest out on a gel and physically cutting out the fragment that you want with a scalpel.

4) Ligation

Once both the vector and the target DNA have been cut we mix them together and add the ligase enzyme. This enzyme ligates (connects) the phosphodiester backbone acting as a glue to stick the ends together.

5) Transform your bacteria

This just means add the DNA back into a bacteria. There are a number of different ways of doing this, but most of them involve making the bacterial cell membrane temporarily porous so that it can take up the liquid containing the DNA mix. Then you let the cells recover and grow them up as usual.

6) Screening for 'positives'; Those bacteria that have the cloned DNA in them.

Sometimes you don't get what you want! For instance, a sometimes time the vector will just stick back to itself, or stick to another vector without having any target DNA in it. So you have to have a mechanism for picking out the ones where the target DNA has gone into the vector.

This is often done using a bacteria that makes a blue colour when fed a particular food but when a bit of target DNA is cloned into it then the gene that produces the blue colour is interrupted so they grow white.

So, you simply pick the white bacteria, grow them up and prepare the plasmid DNA from them, which contains lots of your cloned gene. Voila!

You have to check the white ones out. This is done by isolating pure plasmid DNA (a plasmid mini-prep). Then analysing it by PCR or by use of restriction digests or by DNA sequencing to make sure your gene of interest has be cloned in.

PCR products and restriction digests are analysed by agarose gel electrophoresis.

Source:

https://highschoolbiology.otago.ac.nz/index.php?option=com_content&view=article&id=106

Image: 

https://www.bio.davidson.edu/courses/molbio/molstudents/spring2003/keogh/plasmids.html