Introduction to Biotechnology Methods
Upload a picture of your lab set-up here. Make sure to include your ID in your photo.
Activity 1: Sickle Cell Anemia Detection Simulation
My best advice for completing this section is to have paper and pencil ready and draw out what the DNA fragments look like before and after cleavage. Do not try and do it in your head.
1. The restriction enzyme DdeI produces sticky ends. For the wild-type beta-globin sequence, how many DNA fragments are present in the digestion (cleaving) by Ddel? (Remember to read the sequence from the 5’ to the 3’ end looking for all the places the enzyme will cleave). ______ How many cuts did the restriction enzyme make in total? ________
Wild Type Sequence – 5′ CTG ACT CCT GAG 3′
3′ GAC TGA GGA CTC 5
2. For the mutant beta-globin sequence, how many DNA fragments are present in the digestion (cleaving) by Ddel? (Remember to read the sequence from the 5’ to 3’end looking for all the places the enzyme will cleave). _____ How many cuts did the restriction enzyme make in total? _____
Mutant Sequence – 5′ CTG ACT CCT GTG 3′
3′ GAC TGA GGA CAC 5’
3. Explain why the number of fragments in the mutant type may be different than that of the wild-type beta-globin sequence following Ddel digestion (cleavage).
4. Count the number of base pairs for each fragment. Remember that a base pair includes two bound nucleotides. (the compliments) If a base is not paired because of a sticky end, do not count it. This count determines the approximate size of the fragment.
a. For the Ddel-digested, wild-type beta-globin sequence, how many nucleotides are in each fragment? _____ & _____
b. For the Ddel-digested, mutant beta-globin sequence, how many nucleotides are in each fragment? _____
5. On the basis of fragment size, how can the difference between the wild-type sequence and the homozygous mutant sequence be recognized? ( I posted a video on Sickle Cell Anemia in the laboratory materials section of D2L to help with this answer)
6. What fragments would be present following Ddel digestion of a sample from someone with a heterozygous beta-globin genotype? (The answer is in the video I posted)
7. On the gel diagram, indicate where the DNA fragment(s) in the Ddel-digested beta-globin samples would be expected to run. Draw a line for each band. Use the DNA marker with known base pair sizes to orient the bands. A Ddel-digested sample from a newborn with an unknown beta-globin genotype is drawn in the last lane.
8. Based on the gel diagram, what is the genotype of the unknown sample from the newborn? Explain your answer. Homozygous for Sickle Cell (SS) Homozygous for Normal Hemoglobin (AA) Heterozygous for Sickle Cell (SA)
Activity 2: Mini Gel Electrophoresis of Dyes
9. Draw a diagram of the gel that was loaded with dyes, including which dye was put in each lane. Draw the bands that appeared in each lane following electrophoresis. Indicate the color of each. Which combination of dyes make up each of the two unknown samples?
10. What do the banding patterns in each lane indicate? What do banding patterns represent in a real DNA fingerprint?