Maternal Ancestry Power Point

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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
mtDNA 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’
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’
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 mtDNA from
cheek cells through
saliva collection
http://micro.magnet.fsu.edu/cells/mitochondria/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
•
•
•
•
NaCl (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
Step 4: Tag DNA with probes
• Probes let you “see” what DNA you have
http://www.dnalc.org/resources/animations/pcr.html
Department of Chemistry
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
Cellulose
Absorbent Pad
Membrane Card
Glass Conjugate
Fiber
Cellulose
Immersion Pad
Cellulose
Immersion Pad
Membrane Card
Glass Conjugate
Fiber
Cellulose
Absorbent Pad
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-A13263G
A
G
68
IN
A
G
46
NOT
M-C10400T
C
T
21
IN
C
X-A13966G
T
A
2
G
17
A G
54
NOT
IN
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 (mtDNA)
Gel electrophoresis
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