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

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

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’

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

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

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 Greentiger.com Obtain mtDNA from cheek cells through saliva collection

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

HHMI Howard Hughes Medical Institute Tufts University Department of Chemistry

HHMI Howard Hughes Medical Institute Tufts University Department of Chemistry

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

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

HHMI Howard Hughes Medical Institute Tufts University Department of Chemistry Step 3: Make many copies - PCR Animation of PCR

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

HHMI Howard Hughes Medical Institute Tufts University Department of Chemistry Step 4: Tag DNA with probes Probes let you “see” what DNA you have

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

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

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 IN NOT

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