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Mitochondrial DNA: Where Chemistry, Biology and Anthropology Meet

Mitochondrial DNA: Where Chemistry, Biology and Anthropology Meet. Concept – “From spit to SNP”. Develop an experiment to allow high school and college students to determine their “deep ancestry” using the mtDNA in their saliva. Advantages Engaging for students Real world application

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Mitochondrial DNA: Where Chemistry, Biology and Anthropology Meet

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  1. Mitochondrial DNA: Where Chemistry, Biology and Anthropology Meet

  2. Concept – “From spit to SNP” • Develop an experiment to allow high school and college students to determine their “deep ancestry” using the mtDNA in their saliva. Advantages Engaging for students Real world application Genetics Challenges Keeping cost low Limited time Limited equipment

  3. A A A A A A A G G G T T T T T T C C C T T T A A Single Nucleotide Polymorphsim C 3’ 5’ 3’ 5’ G C 3’ 5’ 3’ 5’ G SNP

  4. Science Background Mitochondria are in all cells Wikipedia - File:Biological_cell.svg Mitochondria contain DNA Wikipedia - Animal_mitochondrion_diagram_en_(edit).svg‎

  5. Science Background Mitochondrial DNA is only inherited from mother; does not recombine

  6. Mitochondrial DNA • Maternally inherited • Several mitochondria per cell • 16,600 bp • Shorter than genomic DNA • No recombination • Higher mutation rate • Obtain mtDNA from cheek cells through saliva collection http://hyperphysics.phy-astr.gsu.edu/hbase/biology/cell.html Greentiger.com http://micro.magnet.fsu.edu/cells/mitochondria/mitochondria.html

  7. Science Background - SNPs • Single Nucleotide Polymorphism • ACCTTGCGCGCTATA • ACCTTGGGCGCTATA • Occur throughout the genome • Nuclear and mitochondrial DNA • Are used as genetic markers Wikipedia - Dna-SNP.svg‎

  8. Step 1: Add lysis buffer Key ingredients: • SDS – breaks cell walls • EDTA – deactivates enzymes in the cell • Proteinase K –digests nucleases which would degrade DNA • Spin – keep liquid

  9. Step 2: Isolate the DNA • NaCl (salt) neutralizes charge on DNA • Add cold ethanol • Centrifuge (spin) • DNA forms white solid

  10. Step 3: Make many copies - PCR • Animation of PCR http://www.dnalc.org/view/15924-Making-many-copies-of-DNA.html

  11. Ligation Reaction

  12. Step 4: Tag DNA with probes • Probes let you “see” what DNA you have http://www.dnalc.org/resources/animations/pcr.html

  13. Lateral Flow Biosensor Creation • Nitrocellulose Membrane- immobilized test zones • Cellulose Immersion Pad- absorb running buffer • Glass Conjugate Fiber- collect sample solution • Cellulose Absorbent Pad- ensure complete flow of running buffer

  14. Colorimetric Readout • Poly-T beads bind to Poly-A tailed probe • Biotin binds to spotted streptavidin • Beads produce colorimetric read-out • Excess Poly-T beads bind to Poly-A control line Positive Negative Image Courtesy of Dr. Ryan Belfour Hayman

  15. Example of Successful Biosensor C-A13263G M-C10400T X-A13966G A G A G C T C T A G A G 68 46 21 2 17 54 IN NOT IN NOT IN NOT

  16. Overview of Experiment Saliva collection PCR (mtDNA) DNA purification Biosensor readout Ligation Gel electrophoresis dehybridize Control line Ligation No ligation “T” “G”

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