Download Niemann Pick LAB

Diagnosing
Niemann Pick
disease, Type C
MODIFIED FROM A SLIDE SHOW
Developed by the
Sanford PROMISE
The Sanford PROMISE
Program for the Midwest Initiative in Science Exploration
The Case
Your summer job is as intern in a genetics lab at a
Mount Blueberry Children’s hospital. A doctor
comes to your team and says that he has a family in
which he suspects three cousins all have NiemannPick type C disease. The family would like to know:
1) Do the children indeed have Niemann-Pick type C?
2) what are the risks of future children in the family
developing the disease ?
Niemann Pick Type C
• Niemann-Pick disease is an
inherited condition in which
patients have abnormal lipid
metabolism causing harmful
amounts of lipids to
accumulate in the spleen,
liver, lungs, bone marrow,
and brain.
• Caused by mutations in
genes NPC1, NPC2, SMPD1
• NPC1 mutations account for
95% of type C cases.
Video of Lysosomal Storage Diseases
Part 1 – Polymerase Chain Reaction
(PCR)
• PCR is a technique used
to amplify specific
regions of DNA
• Start with one molecule
of double stranded
patient DNA and
generate 2 after one
cycle
• Exponential increase in
DNA
Starting
Material
1st cycle
2nd cycle
3rd cycle
PCR movie
http://biology200.gsu.edu/houghton/4564%20'04/figures/lecture%204/pcranimatie.gif
SLIDE FROM SLIDE SHOW BY KIM FOGLIA
PCR primers
• The primers are critical!
– need to know a bit of sequence
to make proper primers
– primers can bracket target
sequence
• start with long piece of DNA &
copy a specified shorter segment
• primers define section of DNA to
be cloned
SLIDE FROM SLIDE SHOW BY KIM FOGLIA
20-30 cycles
3 steps/cycle
30 sec/step
Polymerase Chain Reaction (PCR)
• What is in the PCR
reaction mix?
C
DNA
Sample
PCR Rxn
Mix
G
Thermocycler
A
T
C
A
Polymerase Chain Reaction (PCR)
• What is in the PCR
reaction mix?
C
G
A
T
C
A
Primers
dNTPs
Adenosine
Thymidine
Cytosine
Guanine
DNA Polymerase
Salts and Metals
DNA
Sample
PCR Rxn
Mix
Thermocycler
SLIDE FROM SLIDE SHOW BY KIM FOGLIA
PCR process
• What do you need to do?
– in tube: DNA, DNA polymerase enzyme, primer, nucleotides
– denature DNA: heat (90°C) DNA to separate strands
– anneal DNA: cool to hybridize with primers & build DNA (extension)
What does 90°C
do to our
DNA polymerase?
play DNAi movie
The polymerase problem
• Heat DNA to denature (unwind) it
– 90°C destroys DNA polymerase
– have to add new enzyme every cycle
• almost impractical!
• Need enzyme that can
withstand 90°C…
– Taq polymerase
• from hot springs bacteria
– Thermus aquaticus
SLIDE FROM SLIDE SHOW BY KIM FOGLIA
Kary Mullis
• development of PCR technique
– a copying machine for DNA
1985 | 1993
Part 1 – Polymerase Chain Reaction
(PCR)
Polymerase Chain Reaction
• Step 1: Denature DNA
– Heat it up!
• Step 2: Primer
annealing
– Get the first tracks laid
out
• Step 3: Extension
– DNA polymerase fills in
the gaps
Polymerase Chain Reaction (PCR)
• Cycling Conditions
– Initial Denaturation
• 95˚C for 2 minutes
– Denaturation
• 95˚C for 30 seconds
– Primer Annealing
• 60˚C for 20 seconds
– Extension
• 72˚C for 1 minute
– Final Extension
• 72˚C for 3 minutes
20 Cycles
Part 2 – Family History
Different types of genetic mutations
Punnett Square
T
t
T
t
tt
Niemann Pick Type C
Part 2 – Family History
Part 2 – Family History
The Jones Family History
Part 3 – DNA Electrophoresis
• DNA electrophoresis is
a technique used to
separate DNA by charge
and size
• DNA is a charged
molecule – what
charge?
DNA Electrophoresis
• DNA is separated on an
agarose gel based on
size
• TAE buffer is added to
cover the gel
• A power supply applies
a current across the gel
DNA Electrophoresis
Cathode
(negative)
Anode
(positive)
DNA Ladder
• Where do we expect to
see the DNA bands
from our PCR reaction?
Hypothesis
DNA
Ladder
Affected
Carrier
Unaffected
PATIENT
2000 bp
1500 bp
X
1000 bp
750 bp
500 bp
250 bp
?
DNA Electrophoresis
• Place micropipette tip into TAE buffer
HOVER directly over the well in the agarose gel
• Slowly pipet sample into the well
Sample
Well
Agarose gel
TAE buffer
DNA Visualization
• DNA cannot be visualized
with the naked eye
Visible light
• GelRed will bind to DNA
– GelRed is in the agarose gel
• GelRed is excited by UV light
and will give off visible light
***Dangers of UV light***
UV light source
SET UP THE GELS
1
2
3
4 5
6
7 AC CC UC
8
9 10 11 12 13 AC CC UC
The Jones Family History
#1
#3
#5
#4
#7
#6
#2
Rick #8
#9
#10
#12
Gina #11
#13
Career Pathways
Careers
• DNA Scientist
–
–
–
–
Biomedical lab
Clinical lab
Forensic analysis
Paternity testing
• Clinical Geneticist
Regional Groups
• Identity Genetics Inc.
• Sanford Health
BREAST CANCER GENE TEST
BRCA gene test = blood test that uses DNA analysis to identify harmful mutations in
either one of the two breast cancer susceptibility genes — BRCA1 and BRCA2.
Women with mutations in these genes face a much higher risk of developing breast
and ovarian cancer compared with the general population
Jolie Pitt carries a mutation in the BRCA 1
gene that gives her an 87% chance of
developing breast cancer and a 50% chance of
developing ovarian cancer. She lost her
mother, grandmother, and aunt to cancer.
http://bloximages.chicago2.vip.townnews.com/idahopress.com/content/tncms/assets/v3/editorial/0/e7/0e7962f2-3ba8-11e3-a630-001a4bcf887a/526765798859f.image.jpg
BRCA gene test - not routine/offered only to people who are likely to have an
inherited mutation, based on personal or family history, or who have specific types of
breast cancer.
BRCA gene mutation is uncommon.
Responsible for about 5 % of breast cancers and about 10 -15 % of ovarian cancers.
Genetic counseling= important part of the BRCA gene test process.
Patients learn whether they carry the inherited
BRCA gene mutation and receive an estimate of
personal risk of breast/ovarian cancer.
http://s3.amazonaws.com/nbcf-production-assets/attachments/000/000/385/standard_hires/57e514ad84f3780932dff41d5c63ee2c
SET UP THE GELS
1
2
3
4 5
6
7 AC CC UC
8
9 10 11 12 13 AC CC UC
SET UP PCR TUBES & LOAD NIEMANN PICK GELS
Holly H - #1 Grandpa
Jenny- #2 Grandma /CC
Sangah - #3 Terry / AC
Ally- #4 Lisa / UC
Cole- #5 Becky/ Raelyn #13
Mia- #6 Bobby / Ryan #12
Jesse- #7 Rita / Ryan #12
Tayler- #8 Rick / Gina #11
Sarah- #9 Phil / Paul #10
Jessica- #10 Paul / Phil #9
Mackenzie- #11 Gina / Rick #8
Erica- #12 Ryan / Rita #7
Heidi- #13 Raelyn / Bobby #6
Jameson- Becky #5 / UC
Sydney- Lisa #4 / AC
Blair- Terry #3 / CC
Holly F- Grandma #2
Hannah-Grandpa #1
NO PCR FIRST!
LOAD BRCA/CANCER TEST GELS
Jenny - Healthy breast patient
Holly H - Breast tumor patient
Sangah - Patient blood (unknown)
Ally- Control p53
Cole- DNA standards
Jameson- DNA standards
Sydney- Control p53
Blair- Patient blood (unknown)
Holly F- Breast tumor patient
Hannah-Healthy breast patient