Since my first blog, Dr. Steiner and I have made great progress in our research. Although it was a long and frustrating process, we were finally able to overcome the scientific obstacles we faced while trying to genotype the ten adult zebrafish. The ten zebrafish held in the beginning of the experiment were from Hudspeth’s lab in Rockefeller. These zebrafish carried either a wildtype or heterozygous genotype. However, the genotype of each zebrafish was unknown. We were more interested in discovering which zebrafish carried the heterozygous genotype because that meant they carried the recessive allele for the Robo3 mutation. The techniques we had difficulty receiving significant results from were PCR and Gel Electrophoresis. These two techniques were used to genotype the zebrafish. After altering the procedure time and time again we were finally able to perfect it thus leading to valuable results.
In order to genotype the zebrafish we first had to collect a DNA sample from each individual fish. This is also known as DNA Extraction. To carry out DNA Extraction, we first isolated the zebrafish into separate tanks. We then sedated one zebrafish using an anesthetic to prevent the fish from moving. After it was immobilized, I carefully picked up the zebrafish and cut a piece of its fin using sterile scissors. The fin contained the DNA and was placed into its appropriate tube. The zebrafish was returned to its tank, where it recovered. These steps of DNA Extraction were repeated until a DNA sample was taken from all ten zebrafish. Between each DNA extraction, the scissors were cleaned with alcohol. This was a crucial step in this process because it prevented the DNA samples from being contaminated. These extracts were the template for the PCR reactions.
Polymerase chain reaction (PCR) is a technique used to amplify a single copy or many copies of a piece of DNA. The PCR procedure we created that gave us our desired results was much simpler than all the other procedures we tested and failed. Dr. Stiener and I were amazed to see that we didn’t need a fancy PCR mix or expensive enzyme to make it work. To set up the PCR reactions first we labeled the appropriate number of PCR tubes identical to how the microcentfigue tubes used in DNA Extraction were labeled. We assembled the following reaction in each of the ten PCR tubes by adding 15 µl of PCR Master Mix, 4 µl of Nuclease Free Water, 1 µl of correct DNA sample, 4.8 µl of TWT 209 Forward Primer and 4.8 µl of TWT 209 Reverse Primer. The tubes were lightly mixed and placed into a Thermal PCR Block to run at specific cycles. These cycles ran at particular temperatures for different periods of time to allow the primers to bind and so that the polymerase could extend the primer.
After performing PCR we used a Fok I Digest to cut the DNA strand at a specific site. Fok I is a type II restriction enzyme that recognizes a specific nucleotide sequence and cleaves the DNA strand outside of the recognition site. After creating the Fok I Digest mixture and distributing it into ten tubes, the tubes were left in a heat bath set to 37 Degrees Celsius for 30 minutes. I made an agarose gel using 50mL of TBE Buffer and 0.5g of agarose to use in gel electrophoresis. When the gel solidified, 10 µl of each Fok I Digest tube were loaded in individual wells of the agarose gel. The gel was then left to run at 100 volts for 45 minutes. The gel was then placed under a UV light to better visualize the results.
To examine to the results of the gels, Dr. Steiner and I looked for lanes that contained two bands. Two bands meant the zebrafish was heterozygous and one band meant the zebrafish was wildtype. The results indicated that four out of the ten zebrafihs were heterozygous (2 female and 2 male). This was very exciting news for the both of us because we were so use to failing. To continue our experiment we mated two heterozygous zebrafish. This cross led to 25% of the progeny being homozygous recessive. These offspring express the Robo3 mutation. The next step in our research is to mount three wiltype and three heterozygous zebrafish larvae under the confocal microscope and destroy their interneuromast cells using a powerful laser. After the zebrafish will be left over night and the confocal microscope will take pictures of the internuromast cells regrowth. It has been an amazing experience working with Dr. Steiner so far. I am learning so much from him and am grateful for being given this amazing experience.