• Question: How can scientists put parts of human DNA in a plasmid if it is so small?

    Asked by anon-244767 on 29 Apr 2020.
    • Photo: Leanne Milton-Harris

      Leanne Milton-Harris answered on 29 Apr 2020:

      That’s a great question – it’s something we need to do a lot in the biochemistry lab. The human genome is huge, billions of base-pairs all together but we don’t usually look at the whole thing at once. Instead we focus on little sections of the human DNA, called genes. These are sections of DNA that contain the code for a specific protein – if that bit of the DNA is translated, it tells us what order to fasten the amino acids together to make a protein.

      If we know that one particular gene is important for a disease, we can take just that bit of the DNA. It’s complicated, but we would usually start by finding the messenger RNA that contains the sequence of our gene. This has been processed by the cells so it only has the important bits of the sequence of our gene. Then we use a technique called reverse transcription PCR to convert that RNA back to DNA and make lots of new copies!

      When we have all the copies of our gene sequence, we can do something called cloning to add that piece of DNA into our plasmid. There are lots of ways to do that in the lab, and people are inventing new techniques all the time! The most common way is using proteins called restriction enzymes. They recognise a particular sequence of DNA and cut the bonds holding it together, leaving us with “sticky ends”. If we cut our gene and our plasmid with the same enzymes, they have matching ends. We can then use a different enzyme called a ligase to stick the ends together, basically gluing the gene into the plasmid. In our tube, we’ll have hundreds of copies of our new plasmid, enough to do all sorts of interesting experiments!

    • Photo: Laura Devlin

      Laura Devlin answered on 29 Apr 2020:

      Wow, what a great question! You would think that this wouldn’t be possible, but it is! You are correct, the DNA and plasmids are very small, we can’t see them just with our eyes. But, when were are inserting parts of human DNA into a plasmid, we don’t have to be able to see them to do this. We aren’t physically doing the cutting and pasting using our own hands, instead we utilise enzymes and proteins that biology has already made, to do the job for us!

      Essentially we make lots of copies of the bit of human sequence that we want to add to our plasmid. One way to do this is doing a reaction called ‘polymerase chain reaction’ (PCR). We also have lots of copies of the plasmid that we want to use in a separate container. We then “cut” the DNA and plasmid using the same enzymes, called restriction enzymes, which locate a specific sequence in the DNA or plasmid and make a cut. All we have to do is add the restriction enzymes to the tube of DNA or plasmid and incubate them in a temperature that they work best for cutting! They are our molecular scissors.

      The circular plasmid is now cut open, and the ends of the human sequence have been cut. As the same enzyme has been used they have complementary ends, that can ‘stick’ together.
      Sooo, when we mix together all of the plasmids, and all of the copies of DNA, they want to join to each other, a bit like a jigsaw puzzle, and we add another enzyme, DNA ligase, to stick them together. DNA ligase is like out molecular glue!

      Then you should have lots of plasmids with the human DNA inserted! You can then to lots of different experiments to see whether the plasmid has the DNA sequence inserted properly, in the correct orientation, as sometimes plasmids might join back up in a circle together, before they have DNA in.

      I hope this has answered your question!

    • Photo: Angela Downie

      Angela Downie answered on 30 Apr 2020:

      This is a really good question! As you have probably figured out DNA is way to small for us to see with our naked eyes, and we need very powerful microscopes to be able to see it. It is also so small that we can’t manipulate it with our own hands, but thankfully we have a variety of tools that can help us.
      As other people have said one of the most useful tools we have for manipulating DNA are restriction enzymes. Enzymes are small molecules called proteins that carry out all the different jobs in our cells that need doing, in the case of restriction enzymes this job is chopping up DNA! Originally, restriction enzymes came from bacteria, where they work as a defense mechanism chopping up DNA that they recognize as foreign, meaning it does not belong in the bacterial cell. However as they are so useful to us, nowadays restriction enzymes are made in a lab, and we keep them in tubes ready to go. Each restriction enzyme recognizes a different sequence of DNA and so we can pick the ones that will cut exactly where we want them to. When they cut they leave some extra bits of DNA we call ‘sticky ends’ that we can use to put our DNA into plasmids.

      In order to make it easier to work with DNA we can also create a lot of copies of the sequence we want carrying out a reaction we call Polymerase Chain Reaction or PCR. In this case we use a different enzyme called Polymerase, that uses the DNA as a tempate to make an exact copy of it. We have machines in the lab called thermocyclers that we program so that the polymerase does various cycles of copying the DNA until we have thousands of copies, making it easier to work with the DNA.
      We also have special dyes that stick to DNA and when exposed to UV light make the DNA shine. In this way we can run DNA on a gel and we can see what size it is which can help us make sure we have the right bits of DNA in our sample.

Comments