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 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

SLIDE FROM SLIDE SHOW BY KIM FOGLIA 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 cycles 3 steps/cycle 30 sec/step SLIDE FROM SLIDE SHOW BY KIM FOGLIA

Polymerase Chain Reaction (PCR) What is in the PCR reaction mix? DNA Sample PCR Rxn Mix 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°C do to our DNA 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 | 1993 SLIDE 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

Niemann Pick Type C

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 AffectedCarrierUnaffected 2000 bp 1500 bp 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

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***

Sample gel

SET UP THE GELS LaddErLaddEr LaddErLaddEr LaddErLaddEr LaddErLaddEr

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