Drosophila Genotyping Using PCR

DNA lab
December 20th, 2021
January 10th, 2022

Medya Badakhshani
Nikola Iliev
 Abstract
Using pipettes, Lysis of Drosophila cells, PCR amplification, and separation of PCR
reactions by Electrophoresis were practiced in two lab sessions. As samples, two species of
Drosophila melanogaster were used: a red-eyed sample fly and a white-eyed one. Unfortunately,
no expected result was obtained in any group due to short cycle time.
In the sample picture it was observed that the white-eyed sample stood higher at 704 bp
while the red-eyed sample stood lower at 467 bp.

Introduction

Drosophila melanogaster

Drosophila melanogaster is a species of fly (the taxonomic order Diptera) in the

family Drosophilae. The species is often referred to as the fruit fly or lesser fruit fly, or less
commonly the "vinegar fly" or "pomace fly”.
D. melanogaster was among the first organisms used for genetic analysis, and today it is
one of the most widely used and genetically best-known of all eukaryotic organisms due to its
rapid life cycle, relatively simple genetics with only four pairs of chromosomes, and large
number of offspring per generation (1).
D. melanogaster has a red-brown eye color caused by the presence of two classes of
pigments: pteridines (red), and ommochromes (brown). There are independent pathways for the
synthesis of each (2)(3).

Polymerase chain reaction

Polymerase chain reaction (PCR) is a laboratory technique used to amplify DNA

sequences. The method involves using short DNA sequences called primers to select the portion
of the genome to be amplified. The temperature of the sample is repeatedly raised and lowered to
help a DNA replication enzyme copy the target DNA sequence. The technique can produce a
billion copies of the target sequence in just a few hours (4).
 Material and methods
Materials

Learning pipetting skills:

• Empty tubes, 2 * 2ml, 0,2ml tube
• Tube containing water
• P-10, P-100, and P-1000 micropipettors
• Aerosol-resistant pipette tips
• Rack for 1,5/2ml tubes + 1 rack for 0,2ml tubes
• Gloves

Cell lysis:
• 0,2ml vail containing a wild type fly
• 0,2ml vail containing a white mutant fly
• 2 * 0,2ml vail containing 48 ul fly squishing buffer without prot K
• Lysis and binding buffer 250ul
• 2ml vail containing sterilized milliQ water

PCR:
• 2 vails containing a PCR bead
• 2ml vail containing 15ul primermix P1/P2/P3
• 2ml vail containing 10ul syto-9

Methods

This experiment had 4 different sections which were divided into two lab practicals:
• Learning pipette skills
• Lysis of Drosophila cells
• PCR Amplification
• Separation of PCR Reactions by Electrophoresis

Learning pipetting skills

In order to be able to continue with the rest of the lab, first the knowledge of using
pipettes to transfer liquids from one tube to another with certain amounts, was practiced.
 Wearing gloves, two 2ml tubes and one 0,2ml tube were placed in racks. Then, P-10, P-
100, and P-1000 micropipettors were used to transfer water to these tubes. This was done by
attaching the right pipette tips and turning the numbers to the volume needed. Coming to the first
stop the pipette tip was placed into the water and slowly released as water gets drawn into the
pipette tip. Then, the pipette was brought out of the water and placed into the empty tube and
pressed to the second stop to release all the liquid into the tube. Holding down the button the
pipette is brought out of the tube before releasing in order to prevent any liquids getting drawn
up. The pipette tips were disposed using the button that releases them.

Lysis of Drosophila Melanogaster

In order to isolate Drosophila melanogaster DNA, a proteinase K procedure was used to
destroy and open the cells.

First, the wild-type and mutant fly were transferred to two 0,2ml vails that contained 48ul
fly squishing buffer by placing them above the vail and gently tapping them. The side of the vails
(not cap) were named W and R in addition to the group names in order to prevent confusion later
in the process.

Second, both flies were squished with a 200-µL pipette with clean tips for each fly. After
the flies were squished, the practical instructor added 2ul proteinase K to the tubes. Furthermore,
the tubes were transferred to a thermal cycler which was programmed to incubate the sample at
60ºC for 60 minutes followed by 94ºC for 2 minutes and then cooling to 4 degrees.

PCR amplification of the Drosophila Melanogaster locus

The following liquids were added to two 0,2ml PCR tubes, labeled W and R, in order to
make the PCR reaction mix:

• 7,5 µl deionized water

• 12,5 ul ready to use Taq DNA polymerase master mix (containing dNTp, salts, buffer,
Sybr Green)
• 2µl primer mix
• 4ul Lysate containing the DNA

The tubes were put in a thermal cycler, and the following program was run:
 Denaturation 37 cycles End extension

94°C for 2 min. 94°C for 60 sec. 72°C for 5 min.

55°C for 60 sec.

72°C for 120 sec.

(measure light
fluorescent light)

Electrophoresis - Conducting Agarose Gel Electrophoresis

A 1.0% agarose gel was prepared by adding 0,5 gr of agarose in 50ml 1x TBE solution.
The sample was heated in a microwave until boiled. Afterwards, a number of minutes was waited
until the solution cooled down (hand warm) and 3-5 µl Midori Green was added. In the meantime
the gel holder was prepared by closing both sides with tape and placing the plastic frame for the
slots at the beginning of the gel holder. The solution was poured into the gel holder and the plastic
slot frame was removed after the gel had solidified. TBE solution was poured on top of the get
until the slot holes were invisible.

Moreover, 5µl of 10x Gel Loading Solution was added to the PCR samples.

The DNA marker was added to the first slot by the practical instructor. Then, 25 µl of the
white and red PCR samples were added to the subsequent slots respectively, using a pipette and
carefully inserting the tip into the slots just below water surface by one of the students. The same
process was repeated twice more by two other students.

Subsequently, the cover was placed on the gel holder snapping it onto the electrode
terminals and the plugs were inserted into a power supply that was set to 150V.

The electrophoresis was conducted until the blue visible marker reached ¾ of the gel.

After the electrophoresis was completed, the power supply was disconnected, and the gel
was carefully transferred to the plastic bed of a transilluminator before the plastic hood was closed
and the device was turned on. Lastly, multiple pictured were taken in order to compare different
groups’ results.
 Results

In Lane 1 200bp ladder can be seen. However,

neither of the samples that different groups made showed desired
results. No travel could be seen on both samples. Therefore, a
sample picture (the following picture) was uploaded by the
practical instructor on Blackboard to be used by the students.

Every band is 200bp longer than the previous one. Therefore, the second
slot from the left is 704 bp long and the third row is 467 bp. That concludes that
the left band is for the white-eyed fly and the right one is for the red-eyed fly.

Discussion

Both samples for this experiment failed. That was assumed to be caused by the thermal
cycles not being long enough. The samples were taken out of the thermal cycler by an employee
after only 37 cycles instead of 40 due to the university closing down for the Christmas/New
year’s holidays which seemed to have caused this result issue.

Conclusion

Although the obtained results in the second lab were not satisfactory, in the sample image
the length of the two different samples were completely visible and easily distinguishable. The
longer band belonged to the white-eyed fruit fly and the shorter band was associated with the
red-eyed fruit fly.
 References

1. Wikipedia contributors. (2022, January 18). Drosophila melanogaster. Wikipedia.

https://en.wikipedia.org/wiki/Drosophila_melanogaster

2. Reaume, A. G., Knecht, D. A., & Chovnick, A. (1991). The rosy locus in Drosophila

melanogaster: xanthine dehydrogenase and eye pigments. Genetics, 129(4), 1099–1109.

https://doi.org/10.1093/genetics/129.4.1099

3. Summers, K., Howells, A., & Pyliotis, N. (1982). Biology of Eye Pigmentation in Insects.

Advances in Insect Physiology Volume 16, 119–166.

https://doi.org/10.1016/s0065-2806(08)60153-8

4. Polymerase Chain Reaction (PCR). (n.d.). Genome.Gov.

https://www.genome.gov/genetics-glossary/Polymerase-Chain-Reaction