Gene Identification & Primer Design
Designed primers to isolate and determine amplicon length in fruit flies
Problem Statement
Students were given a random DNA sequence by the instructor to identify
The experiment was run in silico and in vitro.
In Silico
BLAST
The sequence was sent through the BLAST sequencing database to for similar biological sequences. Hippo was the most similar sequence.
FlyBase
With the name of the gene now known, FlyBase was used to retrieve the genome region.
Primer3Plus
Primer3Plus was used to choose the appropriate region to amplify; the resultant primer was twenty base pairs long on the left and right and amplified a 197 long base pair sequence.
Uniprot
In order to learn the function and structure of the protein its name was pasted into the database Uniprot; its location, function, amino acid sequence, and more vital information was derived and used in vivo.
The function of the gene was determined though the 4 database and gene tool platforms to determine the Hippo gene pathway negatively regulates the activity of transcriptional co-activators that inactive a oncoprotein that controls cell proliferation, and apoptosis. This results in uncontrolled tumor growth.
In Vitro
Sample Set-up
To isolate DNA, the sample of Drosophila melanogaster has been taken by anesthetizing the flies
5 male flies and 5 female flies were taken for the result comparison
The samples were prepared for PCR
PCR
The PCR reaction was set up with the mix used for both male sample and female sample
Therefore, the quality of reaction was multiplied by 2x
The PCR reagents were combined and allowed to run
Gel Electrophoresis
A gel was prepared according to the instructor's and lab manual directions
The samples and DNA ladder were taken off ice and added to the gel and allowed to run for the appropriate period of time before being analyzed
Results
The gel did not yield any results due to a missed heating step in the PCR reaction. If the results had yielded good gel, the region's presence would have been able to be confirmed.
Skills Advanced
PCR
Primer Design
Technical writing
Databases
