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