1. What are two functions of DNA?

2. Briefly outline ONE of the following (4 pts):

1. Explain how the chain terminator method is used to sequence DNA.
2. Explain the process of DNA fingerprinting including the role of genetic polymorphisms and restriction endonucleases.
3. Explain how polymerase chain reaction (PCR) works to amplify a sample of DNA.

Chain terminator method (click on Play for Cycle Sequencing at: http://vector.cshl.org/resources/BiologyAnimationLibrary.htm)

• Isolate DNA
• Incubate DNA in presence of enzyme (TAQ polymerase), primers, deoxynucleotides AND labeled DIDEOXYNUCLEOTIDES.
• As DNA replication procedes it will terminate each time a dideoxynucleotide is incorporated into the growing DNA. (Why)?
• Separate subsequent strands of DNA using gel electrophoresis
• Order bands from short to long
• The sequence of separated fragments is the 5'-3' sequence that is complimentary to the original target sequence (3'-5')

DNA fingerprinting, genetic polymorphisms and restriction endonucleases.

• Isolate DNA
• Incubate DNA with restriction endonucleases
• Separate fragments of DNA using gel electrophoresis
• Identify specific DNA fragments using labeled DNA probes that are complimentary to certain sequences of DNA
• Because of genetic polymorphisms (stretches of DNA, often noncoding strands, that are different between people) the number and length of DNA fragments produced by restriction endonucleases will be unique for each individual (A DNA fingerprint)

Polymerase chain reaction (PCR) (click on Play for Polymerase Chain Reaction at: http://vector.cshl.org/resources/BiologyAnimationLibrary.htm)

• Isolate DNA
• Choose primers (two pieces of DNA that are complimentary to the points where you want to amplify. Primers should bracket the DNA to be amplified)
• Denature DNA (94-96oC)
• Anneal Primers (50-65oC)
• Extend Primers (in presence of DNA polymerase)
• Repeat

3. Construct a restriction map for a circular plasmid from the following data:

 Click here for scanned figure

4. Calculate pI for histadine knowing that pK₁ (a -COOH) is 1.8, pK_R is 6.04 and

pK₂ (a -CNH₃⁺) is 9.33.

5. In what order would the amino acids Lys, His and Ala elute from a carboxymethyl (CM) column at pH 6?

6. Refer to the curves below and answer the following questions:

 See curves on your copy of quiz

a. How do you know by looking at the curves if myoglobin or hemoglobin binds O₂ cooperatively? (1 pt)

b. Under which condition, A or B does O₂ have HIGHER affinity for hemoglobin? (1 pt)

3. How does pH affect O₂ binding to hemoglobin? (For example, would condition A be higher or lower pH than condition B)? (1 pt)

1. Describe in detail, one example of how a change in protein structure affects protein function (for example, cooperative binding of O₂ to Hb; allosteric effects; muscle contraction).

B. Diagram a Watson and Crick model of the double helix and discuss how the following evidence supports or fails to support the model:

i. Pheobus Levene showed that each nucleotide of DNA was made up of a phosphate group linked to a deoxyribose sugar, which, in turn, is linked to one of four nitrogenous bases (adenine, guanine, cytosine and thymine).

iii. Table adapted from Erwin Chargaff 's 1949 paper:

iv. Evidence for a phenomenon termed "hydrogen bonding"

1. Watson-Crick model is consistent with structure and composition of nucleotides. The phosphorylated deoxyribose groups form the backbone of the double helix.
2. Franklin's x-ray crystallographic data of B-DNA indicated that the structure was helical. (Did Franklin base conclusion that the phosphate groups were oriented towards the outside of the helix on knowledge of how much water DNA held)?
3. Ratios of [A]/[T] and [G]/[C] approximating 1.0 was consistent with base pairing in Watson-Crick model.
4. Hydrogen bonding between bases explained stability and geometry of molecule.