If there are “CUES” listed within the question, please USE them and UNDERLINE them in your answer!
Chapter 20
1. Use Figure 20.4 to describe how you would go about genetically engineering a bacterium to produce human
epidermal growth factor (EGF), a protein used in treating burns. Also, how might you identify and isolate individual
bacteria that have been successfully transformed? (CUES: DNA ligase, mRNA, cDNA, reverse transcriptase, E.
coli, plasmid, restriction enzyme, recombinant DNA, ampr gene, lacz gene, ampicillin, X-gal)
2. One strand of a DNA molecule has the sequence: 5’-CCTTGACGATCGTTACCG-3’. Draw the other strand. The
restriction enzyme PvuI recognizes the sequence 5’-CGATCG-3’. Indicate where this enzyme will cut this DNA
molecule. Also, identify two types of bonds that are being cleaved by the restriction enzyme. (CUES: phosphate,
sugar, covalent, hydrogen, nitrogenous bases)
3. A researcher is searching for the bacterial clone containing a particular cloned gene. She knows that part of
the nucleotide sequence of the gene is ATGGCTATC. Explain how she might locate the bacteria that contain the
gene. (CUES: nucleic acid probe, complementary, radioactively-labeled nucleotides, nitrocellulose filter, film)
4a. What are some risks to developing genetically-engineered strains of bacteria?
4b. A microbiologist developed a strain of E. coli that were easily killed by sunlight and whose diet required two
unusual amino acids not normally found outside the laboratory. Why would such a bacterium be “low-risk” to use in
genetic engineering projects.
5. The specificity of restriction enzyme recognition can be used to detect mutations. For example, the enzyme
MstII cuts DNA at CCTNAGG, where N is any base. Around the sixth codon in the beta-globin gene is the
sequence CCTGAGG. There are two additional MstII sites on either side of the sixth codon, such that when MstII
is used to cut human DNA in this region, two fragments of 1.15 and 0.20 kilobases are obtained (1 kilobase = 1,000
base pairs). The sickle allele of the beta-globin gene causes sickle-cell anemia when it occurs in the homozygous
state. In this allele, the sequence around the sixth codon is mutated to CCTGTGG.
a. Identify the point mutation that led to the sickle allele.
b. What size (in kilobases) fragment(s) would result when MstII is used to cut DNA with the sickle allele?
c. Sketch and label the patterns of cuts with normal and sickle alleles on a gel.
d. Could an individual have both patterns? Explain.
e. How can this information be used to make a DNA test for these alleles?
6. How is recombinant DNA technology different from genetic recombination that occurs in meiosis? (CUES:
genes from homologous chromosomes, natural process, laboratory process, genes from any organism)
7. Use Figure 20.7 to describe in detail how to amplify DNA by using PCR. (Cues: thermocycler, denature, primer
binding, Taq polymerase, extension, repeated cycles)
8. Many human genes contain introns. Since bacteria cannot excise introns from mRNA, explain how bacteria can
be used to make large quantities of a human protein. (CUES: cDNA, reverse transcriptase, mRNA, exons)
9. Describe each of the following techniques & then describe the role of complementary base pairing in each. The
techniques to be described are Southern blotting, DNA sequencing, and microarray analysis.
(CUES: Southern—electrophoresis, DNA transfer, probe hybridization, complementary binding)
(CUES: DNA sequencing—dideoxynucleotides, chain termination, electrophoresis, DNA polymerase,
complementary binding)
(CUES: Microarrays—mRNA isolation, cDNA, expressed genes, complementary binding)
10. A bacterial plasmid is 100 kb in length. The plasmid DNA was digested to completion with two restriction
enzymes in three separate treatments: EcoRI, HaeIII, and EcoRI + HaeIII (double digest). The fragments were
then separated with electrophoresis, as shown.
Using the circle provided, construct a labeled diagram of the restriction map of the plasmid. Explain how you
developed your map.
11. As shown in Figure 20.4, the lacZ gene encodes an enzyme that can convert the colorless substrate X-gal into a
bright blue product. A plasmid vector contains a modified version of the lacZ gene with multiple restriction sites
within its coding sequence, and a gene for resistance (R) to the antibiotic ampicillin. This vector is used with an E.
coli strain that carries no other functional lacZ gene and that is sensitive (S) to ampicillin. A biologist clones a
wheat gene by inserting it into the multiple cloning site of the lacZ gene in this plasmid vector. As a control to
ensure that the bacterial cells are viable, she also grows some cells that were not transformed with the wheat
gene. Fill in the table with the results of these transformations:
DNA taken up by E. coli
None
Plasmid only (no wheat gene)
Recombinant plasmid
Phenotype for ampicillin
response (R or S)
Phenotype for X-gal (blue
or white)