1. HHMI Howard Hughes Medical Institute Tufts University Department of Chemistry Mitochondrial DNA: Where Chemistry, Biology and Anthropology Meet

2. HHMI Howard Hughes Medical Institute Tufts University Department of Chemistry 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. HHMI Howard Hughes Medical Institute Tufts University Department of Chemistry Single Nucleotide Polymorphsim A T G C A T G CA T G CA T A T A T G CA T G CA T A T SNP 5’ 3’ 5’ 3’

4. HHMI Howard Hughes Medical Institute Tufts University Department of Chemistry Science Background Mitochondria are in all cells Mitochondria contain DNA Wikipedia - File:Biological_cell.svg Wikipedia - Animal_mitochondrion_diagram_en_(edit).svg‎Animal_mitochondrion_diagram_en_(edit).svg

5. HHMI Howard Hughes Medical Institute Tufts University Department of Chemistry Science Background Mitochondrial DNA is only inherited from mother; does not recombine

6. HHMI Howard Hughes Medical Institute Tufts University Department of Chemistry Mitochondrial DNA Maternally inherited Several mitochondria per cell 16,600 bp –Shorter than genomic DNA No recombination Higher mutation rate http://hyperphysics.phy-astr.gsu.edu/hbase/biology/cell.html http://micro.magnet.fsu.edu/cells/mitochondria/mitochondria.html Greentiger.com Obtain mtDNA from cheek cells through saliva collection

7. HHMI Howard Hughes Medical Institute Tufts University Department of Chemistry Science Background - SNPs Single Nucleotide Polymorphism –ACCTTGCGCGCTATA –ACCTTGGGCGCTATA Occur throughout the genome –Nuclear and mitochondrial DNA Are used as genetic markers Wikipedia - Dna-SNP.svg‎Dna-SNP.svg

8. HHMI Howard Hughes Medical Institute Tufts University Department of Chemistry

9. HHMI Howard Hughes Medical Institute Tufts University Department of Chemistry

10. HHMI Howard Hughes Medical Institute Tufts University Department of Chemistry 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

11. HHMI Howard Hughes Medical Institute Tufts University Department of Chemistry Step 2: Isolate the DNA NaCl (salt) neutralizes charge on DNA Add cold ethanol Centrifuge (spin) DNA forms white solid

12. HHMI Howard Hughes Medical Institute Tufts University Department of Chemistry Step 3: Make many copies - PCR Animation of PCR http://www.dnalc.org/view/15924-Making-many-copies-of-DNA.html

13. HHMI Howard Hughes Medical Institute Tufts University Department of Chemistry Ligation Reaction

14. HHMI Howard Hughes Medical Institute Tufts University Department of Chemistry Step 4: Tag DNA with probes Probes let you “see” what DNA you have http://www.dnalc.org/resources/animations/pcr.html

15. HHMI Howard Hughes Medical Institute Tufts University Department of Chemistry 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

16. HHMI Howard Hughes Medical Institute Tufts University Department of Chemistry 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 Image Courtesy of Dr. Ryan Belfour Hayman Positive Negative

17. HHMI Howard Hughes Medical Institute Tufts University Department of Chemistry 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

18. HHMI Howard Hughes Medical Institute Tufts University Department of Chemistry Overview of Experiment Saliva collection DNA purificationPCR (mtDNA) Gel electrophoresisLigationBiosensor readout “T” “G” LigationNo ligation Control line dehybridize