HHMI Howard Hughes Medical Institute Tufts University Department

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

HHMI Tufts University Howard Hughes Medical Institute 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 mt. DNA in their saliva. Advantages Engaging for students Real world application Genetics Challenges Keeping cost low Limited time Limited equipment

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

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

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

HHMI Tufts University Howard Hughes Medical Institute 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 • Obtain mt. DNA from cheek cells through saliva collection http: //micro. magnet. fsu. edu/cells/mitochondria. html Greentiger. com

HHMI Tufts University Howard Hughes Medical Institute 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. svg

HHMI Howard Hughes Medical Institute Tufts University Department of Chemistry

HHMI Howard Hughes Medical Institute Tufts University Department of Chemistry

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

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

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

HHMI Tufts University Howard Hughes Medical Institute 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

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

HHMI Tufts University Howard Hughes Medical Institute Department of Chemistry Example of Successful Biosensor C-A 13263 G A M-C 10400 T G 68 46 IN NOT C T 21 IN C X-A 13966 G T 2 NOT A G 17 IN A G 54 NOT

HHMI Tufts University Howard Hughes Medical Institute Department of Chemistry Overview of Experiment Saliva collection DNA purification Biosensor readout Ligation dehybridize Control line Ligation “T” “G” No ligation PCR (mt. DNA) Gel electrophoresis