Diagnosing Niemann Pick disease, Type C Developed by Sanford PROMISE Slide show by Kelly Riedell Brookings Biology 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 of all have Niemann-Pick type C disease. The family would like to know: 1) Do the children 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 involving lipid metabolism, which is the breakdown, transport, and use of fats and cholesterol in the body. Signs of Niemann-Pick disease type C include severe liver disease, breathing difficulties, developmental delay, seizures, poor muscle tone (dystonia), lack of coordination, problems with feeding, and an inability to move the eyes vertically. Niemann-Pick disease type C usually appears in childhood, although infant and adult onsets are possible. People with this disorder can survive into adulthood. Video of Lysosomal Storage Diseases

Niemann Pick Type C Autosomal Recessive disorder

The Jones Family The doctor has collected DNA from the following patients: 1- Grandpa Jones 2- Grandma Jones 3- Terry Jones (son) 4- Lisa Jones (wife of Terry) 5- Becky Jones (daughter of Terry and Lisa – has the disease) 6- Bobby Jones (son of Terry and Lisa -- has the disease) 7- Rick Robinson (husband of Rita) 8- Phil Robinson (son of Rita and Rick) 9- Paul Jones (son) 10- Gina Jones (ex-wife of Paul) 11- Ryan Jones (non-identical twin son of Gina and Paul – has the disease) 12- Raelyn Jones (non-identical twin daughter of Gina and Paul) 13- Rita Robinson (daughter)

Micropipettes Used for moving volumes of liquid from 0.2-1000μL Push button/ Adjustable knob Finger rest Used for moving volumes of liquid from 0.2-1000μL Adjustable/Fixed settings Tip ejector button Body Volume display NEVER pipette liquids directly ALWAYS USE DISPOSABLE TIP! Tip holder (shaft) NEVER lay micropipetter down on table with liquid in the tip Micropipette tip

Micropipette Setup 2 3 1 1 1 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 10μL 2 1 3 1 1

Micropipette Operation PRACTICE!

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

Polymerase Chain Reaction (PCR) https://image.slidesharecdn.com/techniqueofpolymerasechainreactionpcr-experimentalbiotechnology-130211215640-phpapp01/95/technique-of-polymerase-chain-reaction-pcr-experimental-biotechnology-19-638.jpg?cb=1360619880 Polymerase Chain Reaction (PCR) Step 1: HEAT to DENATURE double-stranded DNA into single strands. Step 2: COOL to ANNEAL the primers to a specific region of DNA. Used to target specific DNA sequences Step 3: EXTEND using Taq polymerase to add nucleotides to template REPEAT CYCLE PCR video

Polymerase Chain Reaction (PCR) What is in the PCR reaction mix? DNA Sample PCR Rxn Mix dNTPs Adenine Thymine Cytosine Guanine DNA Polymerase Salts & Metals Thermocycler

Taq Polymerase Discovered in bacteria (Thermus aquaticus) in a hot spring Can work in very hot conditions that would denature normal proteins (like human DNA Polymerase)

PROTOCOL for PCR Centrifuge DNA sample 1 minute. Mark 0.2 mL PCR tube (tiny yellow) with patient number Transfer 5uL of DNA sample to 0.2mL PCR tube. Add 20uL of PCR reaction mix to DNA sample in PCR tube and pipet up and down to mix.

PROTOCOL for PCR Place PCR tubes in PCR machine Spread out tubes in slots inside machine Close lid Power button on back

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

Gel electrophoresis – + FROM KIM FOGLIA http://www.explorebiology.com/pptAP/2008/BIOTECHNOLOGY/ Gel electrophoresis A method of separating DNA in a gelatin-like material using an electrical field DNA is negatively charged when it’s in an electrical field it moves toward the positive side DNA         – + “swimming through Jello”

DNA Electrophoresis Gel is placed in electrophoresis tank and covered with TAE buffer DNA samples are loaded into wells in gel A power supply applies a current across the gel

Running a gel 1 2 3 fragments of DNA separate out based on size FROM KIM FOGLIA http://www.explorebiology.com/pptAP/2008/BIOTECHNOLOGY/ Running a gel fragments of DNA separate out based on size cut DNA with restriction enzymes 1 2 3 Stain DNA ethidium bromide binds to DNA fluoresces under UV light

DNA Ladder = Standard One lane contains fragments of KNOWN size By comparing bands in sample to ladder DNA approximate sizes can be determined ALSO allows comparison of DNA run on different gels at different times under different conditions

Hypothesis 2000 bp 1500 bp 1000 bp 750 bp 500 bp 250 bp DNA Ladder Affected Unaffected Carrier 2000 bp 1500 bp 1000 bp 750 bp 500 bp 250 bp

Sample gel

LOADING DNA SAMPLES Hover micropipette tip over to top of the well Slowly release the sample into the TAE buffer and let it settle into the well.

LOADING DNA SAMPLES We will run a double gel 20 slots in gel 13 patients + 1 ladder/gel So some lanes will be empty MARK WHICH PATIENT IS RUNNING IN WHICH WELL! LOAD DNA Ladder – 5 uL Patient DNA- 10 uL in each well

DNA is INVISIBLE to the naked eye. Two kinds of dyes will be used to be able to “see” the DNA in the gel. TRACKING DYE- is added to DNA sample. - DOESN’T label DNA bands - Labels the front as the DNA moves down the gel. - Signals when to stop running the gel   GelRed- added to agarose gel - binds to DNA and LABELS DNA BANDS - when excited by UV light it will give off visible light - allows visualization of bands

Part 2 – Family History

Part 2 – Family History

The Jones Family History

STANDARD CURVE LADDER fragments are a KNOWN SIZE GRAPHING THE LADDER PROVIDES A COMPARISON TO DETERMINE APPROXIMATE SIZES OF UNKNOWN SAMPLE FRAGMENTS ALSO ALLOWS COMPARISON OF FRAGMENTS RUN AT DIFFERENT TIMES ON DIFFERENT GELS

STANDARD CURVE If a fragment traveled 26 cm on this gel what size is it? 1000 bp