1. 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

2. 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?

3. 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

4. Niemann Pick Type C Autosomal Recessive disorder

5. 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)

6. Micropipettes Used for moving volumes of liquid from 0.2-1000μL
Push button/
Adjustable knob
Finger rest
Used for moving volumes of liquid from μL
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

7. 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

8. Micropipette Operation
PRACTICE!

9. 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

10. Polymerase Chain Reaction (PCR)
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

11. 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

12. 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)

13. 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.

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

15. 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

16. Gel electrophoresis – +
FROM KIM FOGLIA
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”

17. 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

18. Running a gel 1 2 3 fragments of DNA separate out based on size
FROM KIM FOGLIA
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

19. 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

20. 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

21. Sample gel

22. 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.

23. 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

24. 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

25. Part 2 – Family History

26. Part 2 – Family History

27. The Jones Family History

28. 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

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