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Isolation, Extraction, and Identification of Yellow Body Gene in D. Melanogaster 1 Isolation, Extraction, and Identification of Yellow Body Gene in D. Melanogaster Tarek Abugattas, Sara Larocca, Quiche Colon-Davilla, Amraiz Ocasio University Of South Florida Genetics Lab PCB:3063L Section:002 Jessica Wohlfahrt 02/26/2024

Isolation, Extraction, and Identification of Yellow Body Gene in D. Melanogaster 2 Introduction One of the key components of the evolution of genetics is the ability for genes to mutate. This is the fundamental driving force for evolution on earth as helpful mutations will stay in the species because it is more advantageous to live life with them instead of without. There are four major types of genetic mutations translocation, duplications, inversions, and deletion. Translocation of DNA is when one part of the chromosome, either the beginning, middle, or end, migrates to another chromosome. Duplication occurs when a segment of DNA is duplicated and shows up twice or more on the same chromosome. Inversion occurs when part of the DNA sequence is flipped, causing a miss order in gene expression. Deletion occurs when one or more segments of DNA are excluded from duplication all together. Notoriously the worst of the four types of mutation, deletion causes the most radical changes in phenotypic expression. Observed for over a hundred years there is a mutation in D. Melanogaster that causes the body to turn yellow instead of black. This mutation has been noted to affect their reproduction with wild type females and hinder their survival in the wild (Jonathan H, et al., 2019). In this experiment the topic of focus will be, what DNA mutations are responsible for the mutant yellow body color phenotype in yellow body D. Melanogaster’s. Following the biochemical pathway for body pigmentation there are four locations of interest, the Hemolymph, the Basal, the Apical, and the Cuticle. The decrease of dopamine production in the cuticle layer causes no black melanin to be produced. This mutation then leads to the yellow body color scene in the Drosophila in this experiment. The phenotype of the Drosophila was first analyzed under microscope noting the sex, eye color, type of wings, and body color. The DNA of D. Melanogaster which turns the body yellow instead of black was isolated, extracted, and identified. The purpose of the isolation, extraction, and identification of this gene is to study

Isolation, Extraction, and Identification of Yellow Body Gene in D. Melanogaster 3 what mutation the gene underwent to cause the body to give a phenotype of yellow instead of black. To see the isolated gene the DNA of the sample must first be isolated and extracted. The isolated DNA will then be pipetted into dilutions and mixes depending on the machine’s parameters. For PCR, the mix will include enzymes and primers to ensure the DNA is cleaved and replicated. The PCR machine is to ensure the specific sequence is being isolated. The DNA will then run through gel electrophoresis, which is a machine that uses different charges at either end to separate genes by their size and electronegativity. This shows the distinct sizes of the strands cut by the primers to ensure the DNA fragments are the length that was expected. The gene in questioned is hypothesized to be a loss-of-function mutation that codes for a protein that is non-functional or junk in nature causing the black melanin not to be formed. Usually in Drosophila without the yellow body mutation there is a protein coding for black melanin causing Drosophila to be dark brown or black in the wild. This would conclude that there was a deletion during replication or another type of mutation not allowing the protein to successfully code for the black melanin. The anticipated results are to see a different in between the Materials & Methods Procedures are adapted from MBS Lab Manual (MBS, 2024). Genomic DNA Isolation The provided tube of yellow body flies was anesthetized using fly nap by placing the tube on its side opening the foam and inserting the fly nap apparatus inside for approximately one minute. Once the flies went under the vial was opened and using tweezers ten flies were removed and placed under the microscope were the phenotypes were analyzed. The flies were then transferred to a 1.5ml plastic tube with 200 micro liters of lysis buffer. This buffer creates a way

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Isolation, Extraction, and Identification of Yellow Body Gene in D. Melanogaster 4 to extract an accurate amount of the DNA using a micropipette. The flies were then crushed in the buffer for approximately five minutes using a plastic grinder until little to no visible body parts were remaining. Once the flies were crushed the vial was vortexed for approximately thirty seconds and then centrifuged for a minute. This separated the vial into two distinct layers allowing the extraction of DNA to be possible with minimal contaminants. The top layer containing the DNA was extracted and placed into a new 1.5 ml tube while the body debris was left in the original tube and disposed of in the biohazard waste. The new tube containing the DNA was then placed under the fume hood where 200micro liters of phenol/CHCL3 was added. The new solution was vortexed for about 1 minute allowing the phenol/CHCL3 to denature the proteins and extract the nucleic acids. This solution was then placed with other groups solutions in the centrifuge for five minutes at full speed. Using a micropipette 100microliters were extracted from the top layer into a new clean and labeled 1.5ml tube. The old tube was discarded in the phenol hazard waste under the fume hood. Next the new tube had 4microliters of RNase added to it vortex and incubate at room temp for thirty minutes. These steps were repeated by the other group mates for the wild type flies. Gel Electrophoresis 5microliters of the RNase-treated DNA was pipetted and placed in a new 1.5ml tube. The tube then had 4microlites of water added as well as 1microliter of 10x Gel Loading Dye. This dye allows the sample to be visible to ensure there was DNA in the correct sample wells. PCR The RNase treated DNA tubes were used to create a 1:20 dilution for both mutant and wild type DNA. This was done by adding 95microliters of molecular grade water and 5microliters of DNA. The tubes were properly labeled mutant and wild type, then all 4 tubes

Isolation, Extraction, and Identification of Yellow Body Gene in D. Melanogaster 5 were placed on ice. A master mix was created using 4micrliters of 10microliter Forward Primer, 4micrliters of 10microliter Reverse Primer, 16microliters of TAQ 5x mix, and 48microliters of water. 18microliters of the master mix was placed into the labeled PCR tubes adding 2microliters respectfully of 1:20 dilution wild type DNA, 1:20 dilution of Mutant DNA, and Water into each of the corresponding tubes. The water was used as a control for the PCR to be analyzed against. Then the Cycle was run. Our first cycle did not show results for the mutant gene and it was suspected that phenol was still present in our original undiluted DNA. This was the case as when it was centrifuged there was still phenol present which was successfully extracted under the fume hood. These steps were then repeated adding a fourth tube for a stronger 1:10 dilution to ensure enough DNA was present for the mutant tube for the PCR to run successfully. This caused to adjust our master mix by multiplying the values by five instead of four giving us 5micrliters of 10microliter Forward Primer, 5micrliters of 10microliter Reverse Primer, 20microliters of Taq 5x mix, and 60microliters of water. Then 18microliters of the master mix was placed into the labeled PCR tubes adding 2microliters respectfully of 1:20 dilution wild type DNA, 1:10 dilution of Mutant DNA, 1:20 dilution of Mutant DNA, and Water into each of the corresponding tubes. These were then run by the TA and we are awaiting the results

Isolation, Extraction, and Identification of Yellow Body Gene in D. Melanogaster 6 References Moore, J. Garey, J. University of South Florida, General Genetics Laboratory Manual/Notebook. General Genetics Laboratory Manual. Lindsley, D. L., & Zimm, G. G. (2012). The genome of drosophila melanogaster . Elsevier Science. Jonathan H Massey, Daayun Chung, Igor Siwanowicz, David L Stern, Patricia J Wittkopp (2019) The yellow gene influences Drosophila male mating success through sex comb melanization eLife 8:e49388

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