Diagnosing Niemann Pick disease, Type C
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Transcript of Diagnosing Niemann Pick disease, Type C
Diagnosing Niemann Pick
disease, Type CDeveloped by the Sanford PROMISE
The Sanford PROMISEProgram for the Midwest Initiative in Science Exploration
Lab Safety
• What’s wrong with this picture?
Lab Safety
• What’s wrong with this picture?– Gloves– Goggles– Lab coat– Posture– Work area
• Shower/eyewash• Spills• Emergency exits
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 of all have Niemann-Pick type C disease. The family would like to know:1) the children indeed have Niemann-Pick type C2) 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
Micropipettes• What is a micropipette for?
– Used for moving volumes of liquid from 0.2-1000μL
– Adjustable/Fixed settings• Why should disposable
micropipette tip be used?– To prevent sample and
micropipette contamination
Push button/Adjustable knob
Tip ejector button
Volume display
Micropipette tip
Body
Tip holder (shaft)
Finger rest
Micropipette Setup• Setting the delivery volume– Pull out adjustment knob– Turn to adjust delivery volume– Check volume display while setting
• Reading the volume display– Unique for each
pipette– 20 – 200μL range 2
30
01
1
10μL
Micropipette Operation
PRACTICE!
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
1st cycle 2nd cycle 3rd cycleStartingMaterial
Step 1: Denature the double-stranded DNA into single strands.
Step 2: Anneal the primers to a specific region of DNA.
Step 3: Extend by synthesizing new DNA using the enzyme DNA polymerase which uses the original strand as a template for nucleotide placement.
Part 1 – Polymerase Chain Reaction (PCR)
Part 1 – Polymerase Chain Reaction (PCR)
Polymerase Chain Reaction (PCR)
• What is in the PCR reaction mix?
A
AC
T
GC
DNASample
PCR RxnMix
Thermocycler
Polymerase Chain Reaction (PCR)
• What is in the PCR reaction mix?
PrimersdNTPs
AdenosineThymidineCytosineGuanine
DNA PolymeraseSalts and Metals
A
AC
T
GC
DNASample
PCR RxnMix
Thermocycler
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
Different types of genetic mutations
Part 2 – Family History
Punnett Square
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?
HypothesisDNA
LadderAffected CarrierUnaffected
2000 bp
1500 bp
1000 bp
750 bp
500 bp
250 bp
DNA Electrophoresis
• Place micropipette tip into TAE buffer directly over the well in the agarose gel
• Slowly pipet sample into the well
Well
TAE buffer
Agarose gel
Sample
DNA Visualization
• DNA cannot be visualized with the visible eye
• 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
Visible light
Sample gel
Results
The Jones Family History
Career Pathways
Careers• DNA Scientist– Biomedical lab– Clinical lab– Forensic analysis– Paternity testing
• Clinical Geneticist
Regional Groups• Identity Genetics Inc.
• Sanford Health