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Transcript
Making DNA relevant and exciting
in the high school classroom
By Anna Heyer and Rachel Zenuk
What is BioME?
• Is it like a biome?
2
Bio and Me?
3
Biology from molecules to
evolution?
4
What is NSF’s GK-12 initiative?
• ~100 5-year training grants nationwide
• Funds grad student partnerships with K-12
public school teachers
• Goal: fellows acquire communication and
teaching skills, while enriching STEM
education in K-12 classrooms
• Training a new generation of scientists
• Strengthening partnerships between
universities & local school districts
What is the focus of BioME?
• Focus: teaching the life sciences via
hands-on experiences, using a
framework of evolution &
biodiversity
We are entering the fourth of five years
What’s the goal of a teacher-fellow
partnership?
• Partners work together to develop and use classroom
teaching materials in the life sciences
• Partnerships are year-long, with the fellow in the
classroom every week
• The fellow is a resource with scientific expertise
• The fellow is not an aide, sub, or student teacher
BioME taps
into fellows’
individual
skills and
interests.
Aletris Neilis:
dissecting a
puma (!) at
Hermosa
Montessori
Adriana Racolta:
planting a garden
at Liberty Elementary
Matt Herron: teaching
molecular techniques
at Tucson High
Sweet Water Wetland Project
3rd grade class at White Elementary
9
Liberty Elementary Garden Club
10
2nd Grade Insect Projects
11
Summer Institute
• Getting acquainted with BioME philosophy & aims
• Getting partnerships underway via team time
• Introducing teaching resources
• Discussing traits of successful partnerships
BioME forges strong bonds and lasting partnerships.
Who runs BioME?
Principal Investigators Graduate Coordinator
Judie Bronstein
Kathleen Walker
Barry Roth
Stacey Forsyth
K-12 Coordinator
Mary Bouley
Educational Evaluator
Melissa Page
Administration
Melvin Hall
Our Partnership
15
The Manduca Project
16
AP Science Field Trip
• Goal- to increase rigor and interest in AP
science and chemistry
17
San Pedro River
• Students collected insect larva, turtles, and
other aquatic specimens
18
Bob cats, pumas, and snakes...Oh My!
19
A great trip, but most of the
sleeping happened on the bus
20
DNA Extraction
21
DNA Extraction
• Purpose
– to excite students and “hook” them in on the
first or second day of school
– to teach basic procedures and classroom
management
– to introduce the molecule we will be working
with all year
22
Materials
•
•
•
•
•
Small vials
rubbing alcohol or ethanol 70% or higher
toothpicks
liquid dish soap in water (1:2 soap to water)
1% salt solution
23
Procedures
1. Swish 15 ml of salt water for 1 minute.
2. Pour spit mixture into conical soap
tube
3. Rock back and forth for 2 minutes.
4. Add 5-10mL of chilled ethanol to the
tube.
5. PULL OUT THE DNA!
24
Summarize the procedures in
your own words….
1. Swish with salt
water
2. Mix with soap
3. Add EtOH
4. Twirl with
collection stick
5. Preserve DNA
25
How could use DNA extraction
in your classroom?
• Extensions: create DNA necklaces
• Questions???
26
Microarray Lab
Today’s BIG Idea
• Over the last 30 years many defects in genes
have been linked to cancer, each promising to
be the magic in understanding and curing
cancer.
• We now know cancer is a multistep process,
and accumulation of mutations, or genetic
aberrations, allows a cell to progress to tumor
and malignancy.
Cancer is caused by genetic mutations
DNA
CA AG C T A A C T
Normal gene
CA AG C G AAC T
Single base change
CA AG GCG C T A A C T
Additions
C
T
CA A G A A C T
Deletions
Cancer is caused by genetic mutations
Normal
cell division
Cell Suicide or Apoptosis
Cell damage—
no repair
Cancer
cell division
First
mutation
Second
mutation
Third
mutation
Fourth or
later mutation
Uncontrolled growth
Cancer involves MULTIPLE mutations
Benign tumor cells
grow only locally and
cannot spread by invasion
or metastasis
Malignant cells invade neighboring
tissues, enter blood vessels, and
metastasize to different sites
Time
Mutation
inactivates
suppressor
gene
Cells
proliferate
Mutations
inactivate
DNA repair
genes
Proto-oncogenes mutate More mutations,
to oncogenes
more genetic
instability,
metastatic disease
Oncogenes
Normal cell
Cancer cell
Mutated/damaged oncogene
Normal
genes
regulate
cell growth
Oncogenes
accelerate
cell growth
and division
Tumor Suppressor Genes
Normal
genes
prevent
cancer
Normal cell
Remove or inactivate tumor
suppressor genes
Cancer cell
Damage to
both genes
leads to
cancer
Mutated/inactivated
tumor suppressor genes
DNA Repair Genes
Normal DNA repair
TC GAC
Base pair
mismatch
No cancer
TC T AC
AG C T G
TC TAC
TC T AC
AG C T G
Cancer
AG
TG
AG A T G
No DNA repair
MANY Genes are Implicated in Cancer!
• Every cancer can be attributed to a different set of genetic
aberrations, and different genes are either expressed or not
expressed.
• More than 100 different types of cancer can be found
within specific organs!
This makes cancer treatment tricky…
• Each caner has a different potential of being treated by current
therapies.
• For example, it has been shown cancer cells that lack p53 do
not respond well to radiation therapy, and other non-malignant
cells lacking p53 will progress to malignancy in response to
radiation.
p53 protein
NORMAL cell
Excessive DNA damage
Cell suicide (Apoptosis)
• Thus the treatment itself can cause more cancers!
Discussion Questions
• How do you determine the function of a
gene?
• Which genetic aberrations have been
implicated in cancer?
• What cellular functions are affected (turned
ON or OFF) in cancer cells, and how might
these affect normal cell development?
What is the best way to treat cancer?
• Figure out which genes are mutated and which
genes are expressed or not expressed in the
tissue.
• Gene expression of numerous genes can be
looked at by a new technique called
microarray analysis.
What is microarray analysis?
Microarray analysis uses cDNA
to look at gene expression
DNA
Transcription
mRNA
Reverse
Transcription
Translation
protein
cDNA
40
cDNA is labeled with fluorescent dyes
Microarray analysis shows us which
genes are expressed in cancer
Materials
• Microarray slide -- this slide contains genes
involved in cancer
• Disposable pipette
• cDNA mixture solution -- these cDNAs
were made from normal breast tissue
(attached to blue dye) and breast cancer
tissues (attached to red dye).
• Wash solution
• Color developing reagent
Microarray Slide
Symbol
POL1
GAPdH
HK1
ALDH1
GLUT1
ACTG1
DNASE1
RNASE4
TOP1
BRCA1
PDGFR
CYP1A1
BCL2
LIG1
POL1
APAF1
p53
ZNF84
MUC1
G6PD
TNF
ADH4
DNMT1
POLR2A
MDM2
MMP3
VEGF
ACAT1
MCR4
PDK2
GPB
DUSP1
PRL1
JUN
FOS
RASSF1
RAS
SOS
EGFR
Name
DNA Polymerase
Glyc.Ald.Phos.DeH-ase
Hexokinase 1
AldDeHase
Glucose Transporter 1
Actin, cytoplasmic
Deoxyribonuclease I
Ribonuclease 4
Topoisomerase I
Breast cancer type 1 susceptibility protein
Platelet-derived Growth Factor Receptor
Cytochrome P450 1A1
B-cell lymphoma protein 2
DNA Ligase I
DNA Polymerase Iota
Apoptosis Protease Activating Factor 1
p53 (tumor protein 53)
Zinc Finger Protein 84
Transmembrane Mucin 1
Glu.6-phosp DeH-ase
Tumor necrosis factor
Alcohol Dehydrogenase
DNA Methyltranferase I
RNA Polymerase, subunit 2
MDM2
Matrix Metalloprotease 3 (Stromelysin)
Vascular endothelial growth factor
Acetoacetyl-CoA thiolase
Melanocortin receptor
Pyruvate DeH-ase Kinase
Glycerol Phosphatase Beta
Dual-specificity protein 1
Protein Tyrosine Phosphatase
Jun
Fos
Ras-association domain, family 1 protein
ras
sos
Epithelial Growth Factor Receptor
Function
DNA replication
Kreb Cycle
Glycolysis
Converts retinal to retinoic acid, overexpression confers cyclophos. resistance.
Transports glucose molecules into cells for energy
Microtubule formation, cytoskeleton formation
Degrades DNA
Degrades RNA
Aids in DNA supercoiling
Plays a role in DNA double-strand break repair
Integral membrane receptor that binds PDGF
Drug metabolism
Supresses apoptosis
DNA Ligation during replication/repair
Synthesizes DNA on a template strand
Tumor suppressor- Promotes apoptosis in damaged/ irregular cells
Tumor supressor- induces growth arrest and/or apoptosis
May play a role in transcription regulation
Plays a role in cell adhesion, cell to cell interactions
Metabolism, Provides pentose sugars for nucleic acid synth.
Cytokine, may induce tumor cell death. Deficiencies common in cancer
Alcohol processing
Modifies DNA to make it inaccessible thereby inhibiting transcription
RNA Polymerase synthesizes RNA
Inhibits p53-induced arrest and cell death
Degrades extracellular matrix that anchors cells in place
Growth factor that promotes formation of new blood vessels
Ketone body metabolism
Binds melanocortin; multiple downstream effects
Phosphorylates/inhibits PDH complex
Inhibits glycogen phosphorylase
Dephosphorylates and "resets" MAPK
Stops growth signal cascade from receptor tyrosine kinases
Component of AP-1 transcription factor- activates transcription
Component of AP-1 transcription factor- activates transcription
Inhibits cell cycle progression at the G1-S phase transition
Small G-protein, signaling molecule in transcription activation
Tyrosine-kinase receptor signaling molecule, binds SH3 domains
Binds EGF to promote epithelial cell growth
Procedure
1.
Place the slide onto the paper towel.
2.
Add enough of the cDNA solution to the slide to completely
cover it, but not spill off of the slide.
3.
Let the cDNA hybridize with the microarray slide for 5 minutes.
4.
After the 5 minute incubation of the microarray slide with
cDNA, rinse off the excess cDNA with the microarray wash
solution (in squeeze bottle).
5.
Add color solution, again enough to cover the slide but not spill
over the slide. This solution is toxic so take care to not get it on
you, and wash off of skin immediately. Let the color solution
set for 30 sec, then wash off excess with microarray wash
solution.
6.
Record you data.
Results
Symbol Name
Function
5. GLUT1
Transports glucose molecules into cells for energy
Glucose Transporter 1
Breast cancer type 1 susceptibility
10. BRCA1 protein
13. BCL2
B-cell lymphoma protein 2
Apoptosis Protease Activating
16. APAF1 Factor 1
Plays a role in DNA double-strand break repair
Suppresses apoptosis
Tumor suppressor- Promotes apoptosis in damaged/
irregular cells
Modifies DNA to make it inaccessible thereby
23. DNMT1 DNA Methyltranferase I
inhibiting transcription
Matrix Metalloprotease 3
Degrades extracellular matrix that anchors cells in
26. MMP3 (Stromelysin)
place
Growth factor that promotes formation of new blood
27. VEGF
Vascular endothelial growth factor vessels
Stops growth signal cascade from receptor tyrosine
33. PRL1
Protein Tyrosine Phosphatase
kinases
Ras-association domain, family 1 Inhibits cell cycle progression at the G1-S phase
36. RASSF1 protein
transition
44. GSTP1 Glutathione S-transferase
Helps to inactivate and eliminate some types of toxins
47. MYC
c-Myc Proto-oncogene
Activates transcription of growth-related genes
Discussion
• Microarray analysis shows us which genes are
expresses in normal cells vs. cancer cells.
– Why are some genes expressed in normal cells?
– Why are some genes expressed in cancer cells?
– Why are some genes expressed in both?
Discussion
• Genes expressed in normal cells only are
likely tumor suppressors or DNA repair
genes.
• Genes expressed in cancer cells only are
likely oncogenes.
• Genes expressed in both are present in both
conditions.
THANK YOU!
Questions?
Contact the BIOTECH Project:
Dr. Nadja Anderson
[email protected]
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