hhmi howard hughes medical institute tufts university department of chemistry mitochondrial dna:...

HHMIHoward Hughes Medical Institute

Tufts UniversityDepartment of Chemistry

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.

AdvantagesEngaging for studentsReal world applicationGenetics

ChallengesKeeping cost lowLimited timeLimited equipment



Single Nucleotide Polymorphsim

A

T

G

C

A

T

G

CA

T

G

C A

T

A

T

A

T

G

CA

T

G

C A

T

A

T

SNP

5’

5’

3’

3’

5’

5’

3’

3’



Science Background

Mitochondria are in all cells

Mitochondria contain DNA

Wikipedia - File:Biological_cell.svg

Wikipedia - Animal_mitochondrion_diagram_en_(edit).svg



Science BackgroundMitochondrial DNA is only inherited from mother; does not recombine



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



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



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



Step 2: Isolate the DNA

• NaCl (salt) neutralizes charge on DNA• Add cold ethanol • Centrifuge (spin)• DNA forms white solid



Step 3: Make many copies - PCR

• Animation of PCR

http://www.dnalc.org/view/15924-Making-many-copies-of-DNA.html



Ligation Reaction



Step 4: Tag DNA with probes

• Probes let you “see” what DNA you have

http://www.dnalc.org/resources/animations/pcr.html



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

Cellulose Absorbent Pad

Membrane Card

Glass Conjugate Fiber

Cellulose Immersion Pad

Cellulose Immersion Pad Membrane Card

Glass Conjugate Fiber

Cellulose Absorbent Pad



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



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



Overview of Experiment

Saliva collection DNA purification PCR (mtDNA)

Gel electrophoresisLigationBiosensor readout

“T” “G”

Ligation No ligation

Control line dehybridize

hhmi howard hughes medical institute tufts university department of chemistry mitochondrial dna:...

Documents

dia slide

liquid slide

dna spin

t g ca t g ca t

genomic dna

tag dna

saliva collection slide

dna wikipedia file