For DNA cloning, the sequence of interest is inserted into a circular piece of DNA called a .
Content Reviewers:Rishi Desai, MD, MPH
When you hear the term cloning - you might conjure up images of two versions of yourself, one that’s working all day, while the other parties like a rockstar.
While that might be possible in the future, for the moment cloning really works at the level of copying a piece of DNA - like a gene - many times over.
But this is just as cool and has huge implications.
It basically involves taking a gene from our DNA, inserting it into a plasmid - which is a small, circular bit of bacterial DNA, and then making bacteria multiply that gene - gene replication, and use it to make proteins for us - gene expression!
Ok, so our DNA and plasmid DNA have some things in common - first off, they are both double stranded molecules, with each strand made up of sequences of 4 nucleotides - adenine, or A, guanine, or G, cytosine, or C and thymine, or T - arranged in a specific order, like words in a sentence.
Secondly, to form the double helix, the nucleotides use their bases - A, T, C, G to form hydrogen bonds with bases on the opposing strand.
Bases form bonds according to the rule of “complementary base pairing” - which states that in DNA, A always pairs with T by means of two hydrogen bonds, while C always pairs with G, with three hydrogen bonds.
However, the difference between our DNA and plasmids is that our DNA is organized as 46 linear chromosomes, whereas plasmids are circular in shape - like a molecular DNA necklace.
Now, the first step in DNA cloning is digesting our DNA, which contains the target gene we want to clone, by using restriction enzymes which bind to specific nucleotide sequences, called restriction sites.
There’s a huge number of these restriction enzymes that recognize hundreds of different DNA sequences - so say we used the restriction enzyme ecoRI - which binds to every G A A T T C sequence of DNA, and breaks the DNA between the G and the first A.
We can use this enzyme to cleave both our double stranded DNA, containing the target gene, as well as the plasmid DNA, which is where to want to insert it.
So for a simplified example, let’s say we have a double stranded DNA fragment that looks like this - and remember, the two strands of DNA are antiparallel - one running from a 5’ to a 3’ direction, and the other one from a 3’ to a 5’ direction - a bit like two snakes coiled up together but facing different directions.
5’ A T C G A A T T C A A C A G C G T C G A A T T C 3’ 3’ T A G C T T A A G T T G T C G C A G C T T A A G 5’
The bold parts are the restrictions sites that ecoRI recognizes, and the part in between them is the gene we want to multiply.
Now, when we add some ecoRI to this scenario, it comes in and chomps down hard between the G and the first A on the restriction site on each strand, leaving us with this:
5’ A T C G A A T T C A A C A G C G T C G A A T T C 3’ 3’ T A G C T T A A G T T G T C G C A G C T T A A G 5
Now even though plasmid DNA is circular, bear in mind that the same thing happens when ecoRI recognizes its restriction site over there.
So basically, what we’re left with from our DNA is our target gene, which ends with bits of the restriction sites - we call these “sticky ends”.
And the plasmid, likewise, now has a gap with sticky ends on either side.
This allows the coolest thing to happen - when we take our target gene and its sticky ends, and put it together with a plasmid and its sticky ends, all we need to is add an enzyme called DNA ligase, and the two come together like puzzle pieces!