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Chapter 20
1. The restriction enzyme, HindIII recognizes the sequence 5’-AAGCTT-3’, cutting between the two A’s on
both strands. Draw the double-stranded sequence before and after the enzyme cuts. What type of bonds
are being cleaved by the restriction enzyme? (Cues: active site, complementary shape, phosphate, sugar)
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’. Will this enzyme cut this DNA
molecule? If so then please indicate where.
3. Use Figure 20.4 to describe in detail how to clone a human gene using a bacterial plasmid. (Cues: restriction
enzyme, plasmid, DNA ligase, transformed, recombinant DNA, 1st selection, 2nd selection, ampicillin, X-gal)
4. Use Figure 20.5 to describe in detail how to screen a library for a sequence of interest. (Cues:
nitrocellulose filter, radioactive probe, complementary binding, expose, develop, film)
5. Use Figure 20.7 to describe in detail how to amplify DNA by using PCR. (Cues: thermocycler, denature,
primer binding, Taq polymerase, extension, logarhythmic growth, repeated cycles)
6. 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, northern blotting, RT-PCR, and
microarray analysis. (Cues: Southern: electrophoresis, DNA transfer, probe hybridization, complementary
binding, autoradiography, expose)
DNA sequencing: dideoxynucleotides, chain termination, 2’-carbon, electrophoresis, DNA polymerase,
complementary binding)
northern: electrophoresis, RNA transfer, probe hybridization, complementary binding, autoradiography,
expose)
RT-PCR: mRNA, reverse transcriptase, cDNA, complementary binding
Microarrays: mRNA isolation, cDNA, expressed genes, probe hybridization, complementary binding)
7. Describe how some of the biotechniques we have studied can be used to diagnose diseases.
8. Describe how scientists can bioengineer viruses to deliver proper genes to tissues that have mutated genes.
(Cues: deliver, infect, receptors)
9. Describe how plants are bioengineered?
10. Describe some of the concerns regarding safety and ethics with bioengineering.
REQUIRED QUESTIONS (11 – 14)
11. The following is a single strand of DNA. Find all potential “6-base cutter” restriction sites that would exist
in the actual double stranded DNA molecule. (Hint: make the DNA double-stranded to solve this.)
5’-TAGAATTCGACGGGATCCGGGGGGCATGCAGATCA-3’
12. 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.
a.
Using the circle provided, construct a labeled diagram of the restriction map of the plasmid.
Explain how you developed your map.
b. Describe how:
i. Recombinant DNA technology could be used to insert a gene of interest into a bacterium
ii. Recombinant bacteria could be identified
iii. Expression of the gene of interest could be ensured
c. Discuss how a specific genetically modified organism might provide a benefit for humans and at the
same time pose a threat to a population or ecosystem.
13. 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.
14. A pregnant 30-yr-old woman learns her father has Parkinson’s disease. There is no history of the disease
in her husband. A genetic test that can detect the disease is used on her father, husband, fetus, & on her.
Autoradiographic results after Southern blotting are shown below.
Father
___
Husband
___
Woman
___
Fetus
___
___
___
___
___
___
___
___
___
___
___
___
___
Will the woman develop the disease? Explain.
Will the child develop the disease? Explain.
Can Questions
Read the reference article (only 8 paragraphs) and answer the questions below
Reference article: http://www.the-scientist.com//?articles.view/articleNo/39025/title/New-Method-forReprogramming-Cells/
1. What species of animal cells were used in these experiments?
2. How were the differentiated cells reprogrammed?
3. What 2 assays were done to support the cells were indeed pluripotent?