REVIEW GUIDE: Chapter 4- “Introduction to Studying DNA”
Directions: Answer the following questions on YOUR OWN PAPER and in COMPLETE SENTENCES.
Note: Be sure to look up the answers for the 4-1 DNA study questions (key on-line).
DNA Structure/Replication:
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
What is the “central dogma” of molecular biology?
Who is Rosalind Franklin? What was her contribution to the discovery of DNA?
Who were Watson and Crick? What was their contribution to the discovery of DNA?
What are the four nitrogenous bases in DNA?
What kind of bond holds these bases together?
What makes up the “backbone” of DNA?
What is the name of the bond that holds the “backbone” of DNA together?
What are the 3 parts of a nucleotide?
What kind of primer does primase make?
Why must DNA elongate in the 5’ to 3’ direction?
Answer: DNA Polymerase III can only add a nucleotide to the 3’ end of another nucleotide
11. In the following diagram showing the replication of DNA, label the following items: leading
strand, lagging strand, Okazaki fragment, DNA polymerase, DNA ligase, Helicase,
Primase, single-stranded binding proteins, RNA primer, replication fork, and 5’ and 3’
ends of parental DNA. (use the image that can be found in the powerpoint: DNA
replication found on my web site- resources page)
Gel Electrophoresis:
12. What is the function of each of the following in gel electrophoresis of DNA?
a) Agarose gel:
b) Electric current:
c) "Wells" in the gel:
13. Toward which pole (positive or negative) does DNA migrate when electric current is run
through the gel? Why do the DNA molecules move toward this pole?
14. What would happen to the DNA fragments if you forgot to turn the current off?
15. What would happen to your DNA if you oriented the gel tray in the gel chamber with the
wells sitting next to the positive electrode (red)? EXPLAIN
16. What would happen to your DNA if you turned off the power supply and went to lunch,
leaving your gel sitting in the gel chamber (still in the TAE buffer)?
17. Describe how different sized DNA fragments are separated by the gel matrix.
18. Examine the diagram of a DNA agarose gel below and answer the following questions.
What do the bands in the picture of
the agarose gel represent?
b) Which band(s) traveled the slowest?
WHY?
c) Which band(s) traveled fastest?
WHY?
d) Which end of the gel would be
considered the positive end?
e) Which end of the gel would be
considered the negative end?
a)
19. From the lab where we ran various dye molecules on a gel, what was the charge of the dye
molecules that migrated toward the positive electrode? What was the charge of the dye molecules
that migrated toward the negative electrode?
20. Why is an electric current necessary for separating molecules by gel electrophoresis?
21. Define gel electrophoresis.
22. Describe IN DETAIL the two types of gels used in biotechnology (look in lecture notes).
23. Gel electrophoresis is used to visualize what kind of molecules?
24. When would you want to run a 0.8% agarose gel?
25. When would you want to run a 2-3% agarose gel?
26. List all the supplies you would need to run an agarose gel.
27. What kind of buffers are needed and what is the role of the buffer?
28. Why would you run a DNA ladder on your gel?
29. What is the role of sample loading buffer?
30. What do you use to visualize the DNA in your gel?
31. Why would you want to run an agarose gel? Why are they important?
32. A gel shows us the _____________ of the DNA but it DOES NOT tell us the
_____________ of the DNA which can be done using __________________.
33. The size of the molecular products is determined by:
A.
B.
C.
D.
The intensity of the band
The percentage of agarose gel
Comparison with a molecular weight “ladder”
Being familiar with your gel and “eyeballing” it
34. The concentration of the molecular products is determined by:
A. The intensity of the band
B. The percentage of agarose gel
C. Comparison with a molecular weight “ladder”
D. Being familiar with your gel and “eyeballing” it
35. Buffer is used instead of water when making and running gels because:
A. Buffer enhances the transmission of electric currents in water
B. Buffer is more homogeneous than water
C. Buffers provide the salts necessary to help create the current
Sources of DNA
36. What are some key differences between bacterial DNA and eukaryotic DNA?
37. What are plasmids?
38. What are the two types of bacterial cell cultures and describe each in detail?
39. What is an autoclave?
40. What are two types of containers used for mammalian cell cultures?
41. Why are mammalian cells more challenging to grow in a lab than bacterial cells?
42. What is phenol red?
43. What is the difference between self-renewal and potency/differentiation as it is related to
stem cells?
44. Critical thinking: Why is stem cell research important for science? Why is stem cell research
controversial?
45. What is the difference between totipotent and pluripotent?
PCR and Case Study “Which Little Piggy Went to Market?”
46. Define PCR and what does PCR stand for?
47. What are 3 ways you can denature DNA?
48. What are some advantages of PCR?
49. What reagents do you need in each PCR tube?
50. List and explain what happens at each step of PCR.
51. What is the difference between a positive control and a negative control when performing PCR?
52. Draw 2 cycles of PCR starting with 1 strand of parent DNA.
53. Figure 1: Identification of sex-determining region of Y gene in male pig tissue.
(if you need to review the case study its on-line under resources)
369
246
123
Lane
Lane
Lane
Lane
Lane
Lane
1:
2:
3:
4:
5:
6:
DNA Ladder
Negative Control (PCR reaction – no DNA)
Negative Control (PCR reaction – no primers)
Positive Control (158 bp fragment from boar tissue)
PCR of sow tissue
PCR of sausage with male pig tissue
54. Did the PCR reaction work? How do you know? (discuss one of the controls)
55. Did Sue get the PCR results she was hoping for? Explain
56. Why would you want to run a negative control in a PCR reaction?
57. Discuss DNA concentrations for lane 4 and 6.
58. What is the purpose of the DNA ladder in lane 1?
59. Summarize Sue’s next 2 PCR reactions.
60. What results should she report back to John? Did she help him resolve the problem of determining if his
sausage samples contained boar meat? Explain in detail