The Gene Frequency of the CCR5Delta 32 Allele from a Population in Northeastern OhioJacqueline Makowski, Alexis Shawver, Connor Anderson, Alexandra Fulton, Megan Sheldon and Victoria Soewarna Advisor: Dr. Harry Kestler
Delta 32 Deletion of 32 base pairs in the CCR5 sequence Stemmed from Eastern Europe during the time of the Black Plague Black Plague lasted over 500 years This gave the human body a chance to mutate and adapt to the plague Most commonly found in Caucasians Has not been studied in other races
Procedure 2. Determine what primers would1. Map Primers for CCR5 gene visually indicate Delta 323. Develop protocol for DNA 4. Test subject approval ( & Collegesample collection Internal Review Board approval)5. Presenting study to test subject 6. Collect samples (labeled randomly)7. Extract DNA, PCR, Run a gel 8. Repeat process with improvements• Analyze and Record Data • Fix mistakes (contamination, etc.)9. Continue to test and retestsamples 10. Analyze results
CCr5 Sequence map CCR5 Sequence Around Delta32 MutationPrimers F7 & B8 will generate a 140 base pair PCR product for wild-type and a 108 base pair product Δ32 allele.PCR conditions: (5 minutes at 95° (once) 1 minute at 95° (thirty times) 1 minute at60°2 minutes at 72°10 minutes at 72°,then, stops at 4°) using Pure Taq Ready to go PCR beads.
The effects of Δ32 Amino acids come in groups of three called codons GCA ATC GTA 32 is not divisible by three Everything after the 32 base pair deletion mutation consequently changes For example: THE CAT RAN Remove the “C” in “CAT”
Protocol1. Participants were given 2. Inside of cheek swabbeda sterile swab for one minute3. Swab placed in a tube 4. Tubes were randomlycontaining a lysing agent to labeledextract 6. PCR samples were5. PCR was performed on analyzed by agarose gelthe samples electrophoresis
Results• 45 out of the 50 samples are homozygous wild-type • Normal progressor• 5 out of the 50 samples are heterozygous for Δ32 • Long term non-progressor• 0 out of the 50 samples are homozygous for Δ32 • Less likely to contract HIV
Conclusion• Five out of fifty people were found to be heterozygous for the delta 32 deletion mutation• Five alleles out of one hundred alleles or a gene frequency of 5% was found within the Early College Population
Future Plans• Continue testing for the Δ32 deletion mutation at LCCC• Clone the Δ32 mutation into a plasmid vector to transform hematopoietic stem cells • This could be used for gene therapy to potentially cause all new blood cells to be Δ32.