Diagnosing Niemann Pick

disease, Type CMODIFIED FROM A SLIDE SHOW

Developed by the Sanford PROMISE

The Sanford PROMISEProgram 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 Niemann-Pick 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

1st cycle 2nd cycle 3rd cycleStartingMaterial

SLIDE FROM SLIDE SHOW BY KIM FOGLIA

http://biology200.gsu.edu/houghton/4564%20'04/figures/lecture%204/pcranimatie.gif

PCR movie

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

20-30 cycles3 steps/cycle30 sec/stepSLIDE FROM SLIDE SHOW BY KIM FOGLIA

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

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°Cdo to ourDNA polymerase?

play DNAi movie

SLIDE FROM SLIDE SHOW BY KIM FOGLIA

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

Kary Mullis• development of PCR technique

– a copying machine for DNA

1985 | 1993SLIDE FROM SLIDE SHOW BY KIM FOGLIA

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

Different types of genetic mutations

Part 2 – Family History

Punnett Square

t t

t

t

T

T

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 bufferHOVER 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

SET UP THE GELS

1 2 3 4

LaddEr5 6 7 8 9

LaddEr

LaddEr1 2 3 4 5 6 7 8 9 10 10 11 12 13 11 12 13

LaddEr

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