| Optimizing PCR Processes for Drosophila melanogaster DNA to
Detect Polymorphisms in Milton Mutations |
| Project Proposal |
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This project is based upon the Milton genomic regions in Drosophila melanogaster.
These regions affect the flies’ ability to see. The genomic and cDNA sequences
of Milton are known, and the genomic region is 5200 base pairs in length. We
have several strains of flies with different Milton mutations—wild type with a
normal Milton protein, blind Milton type with a stop codon mutation, and a
strain that has been genetically engineered with the green fluorescent protein
(GFP) gene inthe middle of the Milton gene. We shall design several primers and
optimize a PCR reaction so that we can amplify the Milton DNA from the flies and
observe polymorphisms in the various strains through gel electrophoresis.
Finally, we will perform a BLAST analysis on the gene sequence to investigate
any human homologues of the gene. Understanding more about how certain
mutations in Drosophila compare to human genes can help us progress in the field
of human genetic diseases.
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| PCR Overview |
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| About Milton |
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Milton is a sequence of DNA in Drosophila that governs eye functionality.
Mutations in these areas of the DNA can result in badly functioning or completely
nonfunctional eyes. In this study, we are searching for mutations so severe that
the fly is blind. One of our fly strains has a stop codon inserted in the middle
of the Milton code. This mutation by itself is lethal, so a genomic “rescue”
construct has been inserted into the genome to allow the fly to live. In addition,
a genetic marker known as the gene for Green Flourescent Protein has been attached
for tracking purposes. This marker is 750 base pairs in length and it is supposed
to attach to the Milton protein and track it by turning green. Unfortunately,
the green color has not been apparent.
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| Outside Help |
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Steve Stowers, a researcher at Stanford University, gave us flies with the rescue
constructs and indicated that the stop codon is around base pair 2000 in the
Milton sequence; hence, our primers will replicate DNA around the 2000bp region.
The primers were designed using a program called MacVector; they follow this page.
| Potential Primers (Set A) | |
| Potential Primers (Set B) |
| Potential Primers (Set C) |
| Pictures of Milton Mutations |