Over the course of a week, the six of us isolated warts gene from the rest of the genome to analyse certain mutations in the DNA sequence. Warts, being a part of the warts-hippo pathway, is a kinase that controls the growth and development of cells. This pathway is a network of proteins that control the size and tissues of organs. It also stabilizes cell division and controlled cell death. Research into an area such as this is important since cancer is a disease where cells divide uncontrollably. For our research purposes we made use of drosophila melanogaster, commonly known as fruit flies, given that they share 75% of human disease genes with the human species. To be able to separate the warts gene we needed to do a range of experiments including isolating the DNA from the specimen, running a polymerase chain reaction (PCR), completing gel electrophoresis, doing PCR purification and sequencing DNA. We also did some extra workshops to understand the experiments. This is what we achieved:
THE FIRST STEP, EXTRACTING GENOMIC DNA
To begin with, we extracted genomic DNA from the fruit fly larvae. Genomic DNA needed to be isolated in order for us to perform further experiments over the oncoming days.
This process involved us crushing the larvae in a tube so that the DNA within the specimen could be easily extracted. We made use of mechanical pipettes to transport and bind certain liquids including a pre-lysis buffer, lysis buffer, ethanol, wash buffer and elution buffer. Overall, this multitude of liquids served to alter and disrupt the conditions of the cell, in turn causing the cell membranes to break down and the DNA to be released.
Finally, following these steps, we had successfully recovered purified genomic DNA and went on to copy this segment using PCR.
WHAT IS PCR?
PCR stands for polymerase chain reaction which is essentially the process of replicating a certain part of a DNA sequence. Within our experiment, we were looking at fruit flies and in particular the warts gene which is what we intended to replicate. Using the previously extracted genomic DNA, we applied solutions known as primers to target our select segments. Primers are short fragments of DNA complementary to the unique sequence either side of the DNA segment to be copied. We then went on to copy this sequence.
The PCR reaction implemented to replicate these segments involves a 3-step-cycle done with a machine. First, DNA molecules are separated into single strands as the hydrogen bonds are denatured and broken at the high temperature of 95 degrees celsius. The next step is called annealing and it takes place at 55 degrees celsius. This is where the primers bind to their complementary sequence. Finally, the third step, elongation, occurs at 72 degrees celsius and is where Taq DNA polymerase, an enzyme isolated from a bacterium, binds to the primers and uses the single strand as a template to copy the sequence along.
Ultimately, the aim of this section of our experiment was to achieve a correct replication of our selected warts gene and on the whole, this was successful. It was rather fascinating to learn about the elements of the PCR cycle and even more so to see the unfathomable amount of replicated DNA we had attained. Now that we had a large quantity of DNA, it needed to be separated using gel electrophoresis.
This was performed halfway through the second day at GTAC. This technique is used to separate DNA fragments according to their lengths and size. As DNA is a negatively charged molecule, when it is placed in an electric field the fragments move towards the opposite positive end of the gel. In gel electrophoresis, the molecules to be separated are pushed by an electrical field through a gel that contains small pores. The molecules travel through the pores in the gel at a speed that is inversely related to their lengths. This means that a small DNA molecule will travel a greater distance through the gel than will a larger DNA molecule. In our results, we could visualise that two out of the three PCR reactions were successful and were extremely clear when illuminated using UV light. One had not succeeded at this point. Overall it was extremely pleasing to be able to see the result of all the work we had been doing and we were quite pleased with how much we were able to accomplish in about 2 days!
Our next step took place on the second day of our GTAC experience where we purified the DNA sequences we had extracted from the fruit fly larvae, in order to isolate the specific segment of the warts gene for sequencing. This step is important as it removes any other solutions that were added to the DNA such as buffers and leaves us with the purified segment of DNA that can now be sequenced. To do this we first prepared the sample by adding CPB, this is a buffer that is used to ensure the DNA properly binds to the column and the waste can be removed. The solution is then spun in the centrifuge after which the waste is disposed. Next the DNA and solution was washed using CPE. This isolate the DNA and further remove any other solutions it was previously mixed with. After this the DNA and column is dried and the DNA is mixed with CEB, this is an elution buffer which helps to concentrate the DNA and removes the DNA from the column so it can be collected. This process results in pDNA or our final purified DNA sequences.
This process was really interesting and exciting as it was helping to finalise all of our work and prepare it to be sequenced. It was a very similar process as to when we were extracting the DNA out of the larvae so we were able to apply the skills we had learned from our previous day.
Now comes our final experiment. DNA sequencing is the process of determining the order of nucleotides in DNA. Sequencing DNA would allow for the order of the four bases in nucleotides, Adenine, Cytosine, Guanine and Thymine to be determined. In the experiment, DNA sequencing was important in determining the mutation in the warts gene. In order for the mutation to be determined, the base sequences from the DNA in mutated fly larvae and the DNA in fly larvae that weren’t mutated must be compared. The difference in the base sequence would help determine what caused the mutation in the warts gene. Moreover, the information obtained from sequencing the DNA would be useful in understanding the type of genetic information that is carried in the DNA, which may affect the function of its body.
After the process of purification, DNA sequencing occurred. The DNA sequencing involved determining the volume of pDNA (purified DNA) by assessing the intensity of the bond for each PCR product. Afterwards, the pDNA was added to each of the tubes: one containing forward primer, and one containing reverse primer. The primers were added to help target a specific part of the DNA and reveal its DNA sequence identity. The pDNA was then sent to the AGRF (Australian Genome Research Facility) for the order of bases to be read.
In our first workshop we were working with dissection microscopes to look at wild type fruit flies and then compare them with mutated fruit flies to see the difference in their phenotypes. The different mutations we saw were rosy eyed, white eyed and ebony bodied. From that we were able to identify and explore which genotypes were recessive and which ones were dominant. We also further explored how the mutation would affect further generations and how that would be expressed. Overall, this taught us more about how the warts mutation could be passed down the generations and in which genotypes the mutation would be expressed in the fly.
Our first workshop of day four was working with Tony to help understand bioinformatics and how one small change or mutation in DNA can cause problems in the way proteins form and behave. We first reminded ourselves of the PCR process and how DNA was copied and created as well as how this was used in sequencing. We learned that dideoxynucleotides (base pairs that lack an oxygen molecule so that can’t bind to another base pair) that joined to the end of DNA sequences during the PCR process were used and read to create the final sequence of DNA. Two DNA sequences can then be compared in order to see any mutations that can arise and how this may affect the order of codons and which proteins the ribosomes produce.
Scanning Electron Microscope
Our second workshop on Thursday was working with the electron microscope to identify the warts gene phenotype. We learned how to operate the microscope and took detailed pictures of both control flies and flies with a mutated WARTS gene. It was great to be able to see the effects of the mutation up close and in detail. It was also really exciting and interesting to be able to operate an electron microscope and learn how they work - as none of us had ever done it before.
Tour of the Peter MacCallum Cancer Institute
So to conclude our Thursday, we were given the enlightening experience of visiting the Peter MacCallum Cancer Institute. We found it to be extremely interesting and it has sparked interest in a few of us to look at potential future career pathways. We were able to see the different microscopes that were used and were able to understand why fruit flies and zebrafish were animals that were used to research and conduct experiments. It was fantastic to visualise how our experimental results would be used to help the cancer research and was overall a definite highlight for all of us to finish our week on.
On the whole, we as a group found this week to be incredibly informative in the most fun way imaginable. It taught us plenty of new skills and even proved the real-life applications of these aspects of research.
NADIA: My time at GTAC has been fantastic. For me, this experience has illuminated the appeal to a career in research and in particular cancer research to which I have now taken a great interest in. I really enjoyed the practical experiments as they were all entirely new to me alongside all of the new and impressive equipment we were able to try out over the week. I also found the tour of the Peter MacCallum Cancer Institute to be exceptionally insightful since it was a chance to not only further understand some of the research we had briefly looked at but to see the work in action. All in all, I am extremely grateful to have had the unique opportunity of doing a work experience placement at such an inspiring centre surrounded by some truly passionate people, to make friends with other like-minded students and to explore what may be my future.
ZOE: My experience at GTAC was really fun. I experienced what it’s like to work at a research centre and working on cancer. I found it interesting how long it takes to isolate the warts gene, and the percentage of genes we share with flies. I learned how to use many different kinds of equipment, like PCR machines and different types of microscopes. Furthermore, I learnt the difference between the phenotypes of mutated and wild type flies. Also I understand more about how researchers try to find cures for cancer. I am really excited that I got to learn so much about research jobs, since I want to work at a research centre in the future. I am grateful for the opportunity to do my work experience at GTAC, not only did I learn so many new things, but I also met many people who share the same passion for science as I do.
MADELEINE: Throughout my week at GTAC, I enjoyed engaging in different activities. Before this week, I didn’t know much about how scientific research is carried out. The activities and experiments gave me more of an insight about scientific research, more specifically biological research. It was fascinating knowing that the experiments we did contributes to authentic research projects. Moreover, I found it interesting being in different environments and using equipment that I haven’t used before. Some of my highlights included using the electron microscope for the first time, touring around the Peter MacCallum Cancer Centre and performing experiments. In addition, I enjoyed working in a team with like-minded individuals, where we could exchange our ideas and perspectives. Overall, I found the whole experience informative and it was great being able to participate in the program. The experience has definitely made me consider scientific research in the future.
YATHIKSHA: Overall, my week at GTAC has been truly amazing. Personally this week has enlightened me to the various kinds of research that scientists are progressing on throughout many years of their lives, thus creating a personal spark of interest for me in especially cancer research which I have found to have a keen interest in. I have utterly enjoyed using the various piece of equipment which I never imagined that I would actually be able to use and that was definitely extremely impressive. Furthermore, I thoroughly enjoyed the tour around the Peter MacCallum Cancer Institute which was fantastic to see that the work we had done had helped them in a way and gave me an in depth view to understand some of their research they were conducting.. To conclude, I am definitely honoured to have been part of the unique opportunity to complete my work experience at an enthusiastic centre with people who are truly dedicated and meet like-minded students across different schools who have made this week truly amazing.
JOHAN: The experience I had at GTAC was one of great interest and curiosity, and great enjoyment. I found it, although exhausting, wonderful and rewarding to know that the work I and my friends and companions did will help contribute to cancer research. I found that the work subverted my expectations. I was excited at the beginning, and throughout the week my excitement has not lessened one bit. I have met the most fascinating, the cleverest, and the most wonderful people, and have held them in the highest regard. I have found the teachers and scientists here helpful. I also enjoyed the Peter MacCallum Cancer Centre immensely. Overall, my time here at GTAC has been fantastic.
SAVARNA: Overall, I’ve really enjoyed my experience at GTAC. I wasn’t sure what to expect when starting the week, but it's been really interesting and a really valuable experience. I’ve really enjoyed learning about and being able to use technical equipment as well as being introduced to the process used in DNA extraction and sequencing. I think one of the best parts of this week was being able to hear the different pathways that all the scientists helping us in our experiments went through and being able to learn more about their area of study. Being able to talk to people that have expertise in a range of areas allowed me to extend my knowledge and interests in Science and contemplate my own potential career paths. Another highlight of my week at GTAC was touring the peter MacCallum Cancer Centre. It was great to be able to see where the results of our experiments would be used and link what we had learned throughout the week to practical research. Overall, I’ve really enjoyed my week at GTAC, learned so much and was able to contribute to new research that will hopefully help scientists and doctors in the future.