Sequencing DNA requires several steps: first it must be isolated from the cells, thenby PCR, checked using gel electrophoresis, and finally sequenced. Teacher Andrea Shaw became an expert whilst working at NIWA for a year.
Mrs Andrea Shaw (New Zealand Science, Mathematics & Technology Teacher Fellow, 2006)
I’m going to start with a female, and I’m going to take a sharp scalpel and try and make an incision. Shark skin is very tough.
I make two lines, using sterilised tweezers, and then try and cut down to the bone and try to get the skin off as much as possible. It’s quite tough. Then I simply pop it into this little plastic vessel and fill it up with 90% ethanol [to preserve it].
That’s my little sample that I will then take up to the DNA laboratory, and start the DNA extraction process.
So what we’re going to do is take the samples that we’ve collected from the wet lab and cut them into a very small amount – we only need a little bit – and put them into one of these, which is an epi [eppendorf tube].
To that I’m going to add 750 µl [0.75 ml] of this extraction buffer and 20 µl of an enzyme called Proteinase K, mush it up and down a little bit, and then it’s going to sit in the incubator.
Then I come back tomorrow and do the next step, which is a whole lot of chemical processes, putting chemicals in, using the centrifuge, and extracting off the supernatant until I end up with a piece of DNA.
We then go into the amplification process. This is where we put our samples with certain chemicals, and they go through a 30-cycle process of heating and cooling and the DNA sample splits and re-forms so you get multiple copies of the particular gene that we’re interested in. [This process is called PCR].
We then check it to make sure that we’ve got the DNA in the right spot by running it through gel electrophoresis, through a trans-illuminator, which is a machine which basically takes a photo, which I can then use to send on to Korea.
That comes back to me via computer. I put it into a programme called BioEdit, and you can see the different DNA of the CO1 gene for all these different animals. They’ve lumped together quite nicely, with the different species being very similar.