BCH5425 Molecular Biology and Biotechnology

Spring 1999

1. In the follow diagram is an experiment like that which was performed by O.T. Avery. Answer the following questions: (8 points total)

a) What is the fate each mouse?

b) Will viable Pneumococcus be isolated from each mouse? If so, what type will they be, and what will be their physical appearance?

2. Draw the structure of dideoxy cytidine diphosphate. Include the numbering for all atoms of the base, ribose and phosphate(s). (8 points)

3. Draw the deoxydinucleotide: 5' GT 3' Include a 5' monophosphate group. (8 points)

4. If might be possible for the two purine bases, A and G to hydrogen bond in a similar fashion as an A T base pair. However, this arrangement would result in a DNA molecule with structural features or consequences that would disagree with two facts known about DNA. Draw the hydrogen bond arrangement for an A-G base pairing (5 points). List the two facts about the DNA molecule that would not agree with such a basepairing? (5 points)

5. Restriction Enzymes:

Answer the following by providing one example for each: (12 points)

a. This enzyme would be most useful for isolating large DNA fragments

b. This enzyme leaves blunt ends

c. This enzyme cuts once about every 1000 base pairs of DNA

d. The fragments from this enzyme digest cannot be made blunt by "filling in" with DNA polymerase

e. Some fragments from this enzyme digest may not be able to re-ligate with each other

f. This enzyme is useful for Restriction Fragment Length Polymorphism (RFLP) analysis of DNA

6. For the following restriction endonuclease recognition sequences indicate which ones could potentially be protected by dam methylase, dcm methylase, or neither (12 points)

7. For the following oligonucleotides indicate the expected product after treatment with the following DNA modifying enzymes. Indicate clearly if an end is blunt or ragged after treatment. Note: all overhangs are considered to be of indeterminate length. (15 points)

a. E. coli DNA pol I, dNTP's

b. Nuclease Bal-31

c. Mung Bean Nuclease

8. For the following two DNA fragments: indicate how you would treat the DNA to be able to ligate fragment B to A, and at the same time minimize self-ligation of fragment A. Note: overhangs are ~100 basepairs in length Note: all overhangs are considered to be of indeterminate length (10 points)

9. Draw a replication fork during DNA replication showing the detail of lagging strand synthesis. Indicate and identify all enzymes and transient structures that are required to produce a continuous oligonucleotide on the lagging strand. Note: there is no need to diagram or describe how replication is initiated, or show the leading strand synthesis. (9 points)

10. A certain plasmid contains 6,324 basepairs. At physiological pH and NaCl it prefers to adopt 10.6 bp/turn. The plasmid is observed to have a writhe value of +19.4.

a) What is the value for the Twist and Linkage parameters? (4 points)

b) The DNA is placed into a solution of higher NaCl where it prefers to adopt 11.1 base pairs per turn. What are the new values for the Twist and Writhe parameters? (4 points)