1. Bellwork 2-17-2014 1. Look at the picture: 1. Which fragment, A, B, or C is smallest? 2. Which fragment is largest? 3. What is the charge of DNA? 4. Based only on your observations…which band, A, B, or C has the largest quantity DNA?

2. AIMS

3. In the lab Go to the lab and set up your gel box. The flat side of the black wedge should face inwards. The comb must go on the negative (black side).  This is different from how we set it up last time! Raise your hand. A biotechnology student will check your set-up and pour your gel when you group is ready COMBCOMB

4. Patient Medical test We have decided to test the patient and her family for Huntington’s disease based on our research and the patient’s symptoms.

5. While the agarose hardens…. Problem (write on your lab paper)- Who in the family carries the gene for Huntington’s disease? You have taken a DNA sample from each member of the family: father, mother, daughter, and son.

6. How can we use these electrodes? What is DNA’s charge? If DNA is negative, what happens when we turn the electricity on?

7. How does gel electrophoresis work?

8. What is Huntington’s disease? The defect responsible for HD is a small sequence of DNA on chromosome 4 in which several base pairs are repeated many, many times. The normal gene has three DNA bases, composed of the sequence CAG. Healthy gene has 10-24 CAG repeats Huntington’s gene has 37-80 CAG repeats Will a person with a trait for Huntington’s have a thicker fragment on the gel or a thinner one?

9. Who will get Huntington’s disease? Huntington’s disease is a dominant disorder that must be passed down from parent to child What does it mean to be a dominant trait?

10. Since Huntington’s disease is dominant….. You only need to inherit one bad copy from one parent to develop the disease. (remember that you get ½ your DNA from your mother and ½ from you father)) If a trait is recessive how many copies would you need? 2

11. Possible results– copy the image in your notes! Since Huntington’s disease has many more repeats than the normal genotypes, what would you expect to see on the gel if someone has Huntington’s disease vs. someone who does not? Remember, there are two traits for the disease so individuals can have three different combinations: Normal Huntington’s 2 recessive traits (smaller) 1 dominant and one recessive (one large, one small) 2 dominant traits (both large) hh - normal Hh – huntington’s HH – huntinton’s

12. Write your hypothesis: Choose a family member(s)… Option 1: If _______ has Huntington's disease, then their gel will have _______ band(s) [describe what the bands will look like]. Option 2: If _______ does not have Huntington's then their gel will have _______ band(s) [describe what their bands will look like].

13. Make a record of what goes where!! 1. Draw a picture of your gel and indicate where each sample will go (remember there are 8 wells and 5 samples, one sample is a control). MW M D A B 1 2 3 4 5 Daughter Son

14. Observations (10 total) Quantitative (numbers) Qualitative (not numbers) **I suggest someone in your group take a picture on their phone a few times during the process (especially at the end!)

15. Pipettor/Gel Loading Refresher Do not cross contaminate – one sample per pipette tip and discard in the beaker!!! Push all the way down, place in sample, release Push all the way down to release sample into well Don’t scrap the gel, steady hands!

16. Load and run YOUR gels!! 1. Use the yellow pipettor with a fresh tip to load each sample exactly according to your lane map. 2. Raise your hand to have a biotech student add the buffer when you are done 3. Connect cables and run the gel at 125 volts for 15 minutes. Note the starting time. 4. While you wait, make observations and lab notes. (Note any spills or over flow. Did your pipettor tip puncture the gel? Can you see any thing happen in the box?) 5. While you wait, make another diagram of your gel on which to draw your final results. Make it large enough to record measurements.

17. How can you see if someone has the gene for Huntington’s disease? A machine is used to copy the DNA at specific locations. This machine is called a PCR machine. It allows you to make thousands of copies of a short section of DNA (like a gene) so you can visualize and measure the length of the DNA on a gel. =

18. A review of protein synthesis and its application in HD

19. Remember the central dogma of biology

20. What amino acid do these codons code for? AUG CAU GUG GAG UAG

21. What does the HD gene do? The short answer- It is unclear. Researchers are still trying to understand what the Huntington gene does. The gene is essential for development, and absence of the gene is lethal in mice Wild type protein (this is the non- disease type)

22. Why does this mutation make you sick? Over time the mutated gene is copied into long proteins with extra glutamine amino acids. These fragments can then mis-fold and “stick together” in a process called protein aggregation.

23. Why do you think it would be harmful to have proteins sticking together?

24. Results The mom & daughter have bands that are very far apart, indicating one very large allele. The father & son have two bands that are very close together, indicating two healthy alleles with slightly different number of repeats. I just used beads to show my students.

25. Conclusion Questions: Write in paragraph form… 1. Restate your hypothesis. 2. What conclusions can you make about EACH person based on the gel? Give a detailed description of what each person’s gel bands looked like and why they have the disease or why not. Does this support or reject your hypothesis? 3. Based on the patient’s (mom’s) back story, who do you think she inherited the deadly allele from? Why?