DNA cloning

Definitions & Key takeaways

DNA cloning is the process of making multiple copies of a piece of DNA, like a gene. This can be done in many ways, including inserting a target gene into a circular piece of DNA called a plasmid. The plasmid is then transferred into the bacteria in a process known as transformation, and bacteria carrying the plasmid are then selectively grown, making multiple copies of the chosen gene or even synthesizing its encoded protein.

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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.
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: 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!
This new hybrid DNA is called recombinant DNA. Now, just something to bear in mind - along with our target gene, a gene for antibiotic resistance is also inserted into the plasmid.
This will come in handy later on, when we’ll want to selectively grow only bacteria that contain our recombinant plasmid.
Speaking of which - let’s get that plasmid inside the bacteria! For this, we’ll need to put the plasmids and a fresh lot of, say, E.
coli bacteria in the same test tube. Then, a type of environmental stressor, like a heat shock, is applied and this makes the E.coli membrane more permeable to the plasmids.
However, not all the bacteria respond well to this kind of treatment, so while some of them incorporate the plasmids, others don’t.
In order to separate out the population of bacteria that has incorporated the plasmid and therefore contains our target gene, we take them out of the test tube, and move them on a brand new agar plate to grow them.
This plate has all the nutrients E. coli needs to grow and multiply, but with a twist - it also has some antibiotic sprinkled in there.
Bacteria which haven’t incorporated the plasmid lack the antibiotic resistance gene, so they are virtually helpless in the face of antibiotics - and die off.
On the other hand, those which were clever enough to allow a plasmid inside carry the protective gene which allows them to multiply in spite of the antibiotic.
As these bacteria divide, they replicate their DNA, and also the plasmid - so each time the number of bacteria doubles, so does the number of plasmids, and, in turn, our target gene.
But, remember - genes code for proteins. So basically, what this means is that we can use the bacteria as protein “factories”, meaning they transcribe our gene along with their own, and make proteins that we can then extract and use - for example, let’s say our target gene was the CFTR gene - which codes for a protein called cystic fibrosis transmembrane conductance regulator - a chloride channel that shuttles chloride ions in and out of the cells.
This gene is mutated in individuals with cystic fibrosis, so they produce either too little of this protein, or an abnormally shaped one.
Whichever the case, it can’t do its job right, so patients typically have problems with their pancreas and their lungs - and while we may be some way from a genetic cure for cystic fibrosis, some studies have shown that by using a vector to incorporate the normal proteins and then administering them to patients, it can improve lung function.
Alright, as a quick recap… DNA cloning refers to multiplying small bits of DNA, like genes, and it uses small bits of circular bacterial DNA called plasmids.
The first step 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.
The same restriction enzyme is used to cleave both our DNA, containing the target gene, as well as the plasmid DNA. This results in a target gene with sticky ends, and a gap in the circular plasmid DNA, which join together like puzzle pieces when DNA ligase is added to the mix, and form recombinant DNA.
Then, the plasmids are transferred into E. coli bacteria, and the bacteria are selectively grown, to produce multiple copies of the target gene, or its protein product.