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GUIDED READING - Ch. 20 - BIOTECHNOLOGY
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NAME: ________________________
Please print out these pages and HANDWRITE the answers directly on the printouts. Typed work or answers on
separate sheets of paper will not be accepted.
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READ FOR UNDERSTANDING and not merely to complete an assignment. Though all the answers are in your textbook,
you should try to put answers in your own words, maintaining accuracy and the proper use of terminology, rather than blindly copying the
textbook whenever possible.
1. Define the following terms.
a.
Recombinant DNA:
b. Biotechnology
c.
Genetic Engineering:
DNA cloning yields multiple copies of a gene or other DNA segment. [2]
2. Plasmids are important in biotechnology. Give a full and complete definition of plasmid. [2]
3. What is the difference between the terms DNA cloning and gene cloning?
4. What is a Recombinant Bacteria?
5. a. Using Figure 20.2, label and explain the four steps in this preview of gene cloning.
1.
2.
3.
4.
b. What are two broad uses of gene cloning? Include two examples of each. [2]
1.
2.
6. a.
What is a ‘restriction enzyme’?
b. What was their original purpose in bacteria?
c.
What is the other name for a restriction enzyme? [2]
7. Define the following terms.
a.
Restriction site:
b. Restriction fragment:
c.
Sticky end (and their usefulness):
d. DNA ligase:
8. Explain the steps for making recombinant DNA using a restriction enzyme and DNA ligase by labeling below.
9. What is a Cloning Vector? [2]
10. a. When cloning DNA using bacterial plasmids, we must be able to identify the transformed bacteria. Which
two genes are often included on engineered plasmids used for cloning AND what properties do they impart
bacteria with. Make sure you write the name of the gene correctly.
1.
2.
11. Clones of genes can be used in sequencing the gene, producing the protein the gene codes for, research
and other applications. Label the figure below illustrating the process of cloning eukaryotic genes in bacteria.
12. Now in words….Describe the five-step process of cloning a human gene in bacterial plasmids.
1.
2.
3.
4.
5.
13. Let’s review…
a.
Many bacteria have proteins in their plasma membrane that allow them to take up foreign pieces of DNA. E
coli does not but it still can be induced to take up foreign pieces by exposing the bacteria to a bit of
electricity, called electroporation, or a Ca2+ containing solution. After transformation, how do scientists
know any plasmid, recombinant or not, has been taken up by the bacteria?
b. If the medium used did not contain ampicillin, what other colonies would grow? [1]
c.
What colors would they be? [1]
d. Where is the restriction site conveniently located in your cloning vector?
e.
In your own words, detail how scientists distinguish between the resulting bacterial clones that carry
RECOMBINANT plasmids, plasmids carrying the DNA you wish to clone, and those that do not.
14. The cloning procedure described in question 11 and Figure 20.4 will produce many different fragments of
hummingbird DNA that could be stored now in a genomic library. What is the purpose of a genomic library? [2]
15. Compare a ‘genomic library’ to a ‘cDNA library’. Be sure to define every term used, describe how both libraries
are made, and explain the advantages of each.
a.
b.
16. a. How are bacteriophages used for making genomic libraries?
b. What are the advantages of this? [2]
17. a. Explain how Bacterial Artificial Chromosome (BAC) libraries are formed. [2]
b. What advantage does using BACs as vectors provide?
18. a.
Once all our hummingbird DNA fragments are cloned, we have the problem of finding the DNA piece that
holds our gene of interest. Explain how Nucleic Acid Hybridization will accomplish this task. [2]
b.
What is a nucleic acid probe?
c.
Why do scientists use a radioactive isotope or fluorescent tags for the probes? [2]
d. Describe how a radioactively labeled nucleic acid probe can locate the gene of interest on a multiwell
plate. Use Figure 20.7 to guide your response. [2]
e.
Label the illustration below showing the process of nucleic acid hybridization.
19. Using the information discussed in class, what is a ‘colony blot’ and why is it used?
20. Using the knowledge you have accumulated so far in this course, please explain how DNA denaturation
differs from protein denaturation? [3]
21. a.
What is an ‘expression vector’?
b. What problem does it solve? [3]
22. a. What are two problems with bacterial gene expression systems? [2]
b. What is a ‘yeast artificial chromosome’?
c.
Why would a molecular biologist use yeast as opposed to bacteria for expressing eukaryotic genes of
interest? [3]
23. What is ‘electroporation’?
24. a. Why is polymerase chain reaction such an important tool in biotechnology?
b. The polymerase chain reaction (PCR) is a Nobel Prize–winning idea that is used by scientists to amplify
DNA, particularly when the quantity of DNA is very small or contaminated. Explain the three initial steps
that occur in cycle 1 of PCR. [2]
c. How many molecules will be produced by four PCR cycles? [2]
25. Using your knowledge of DNA’s structure, state the name of the bond hydrolyzed or cleaved by restriction
enzymes? [1]
26. a.
Imagine you have the restriction enzyme PvuI whose restriction site is as follows:
5’ – C G A T C G – 3’
3’ – G C T A G C – 5’
One strand of DNA has the following sequence 5’-CAGGCTAAGCTTGCTACCG-3’. Draw the other strand of
DNA and determine if PvuI will cut this molecule. Why or why not?
b. One strand of DNA has the following sequence 5’-CAGGCGACGATCGTACCG-3’. Draw the other strand of
DNA and determine if PvuI will cut this molecule. Why or why not?
c.
What are the products of the digestion?
d. You want to amplify the double stranded DNA fragment you drew in 26.b using PCR. What would the
sequence be of your two primers if each was 5 nucleotides long? Be sure to also indicate the 5’ and the 3’
end of each primer!!!
DNA technology allows us to study the sequence, expression and function of a gene. [2]
26. a.
What is gel electrophoresis used for?
b. Explain the general procedure for running a gel.
c.
Why is the DNA sample separate by gel electrophoresis always loaded at the cathode or NEGATIVE end of
the power source? [2]
d. Explain why shorter DNA molecules travel father down the gel than larger molecules. [2]
e.
27. a.
Label the illustration on ‘gel electrophoresis’ below.
What is ‘restriction fragment analysis’?
b. To the right of the !-globin alleles, draw a gel showing the different pattern obtained from a normal patient
and a sickle-cell patient after digestion with DdeI. For help, examine Figure 20.10. [2]
c.
How is this procedure used in the diagnosis of certain genetic diseases?
28. A patient who is a carrier for sickle-cell anemia would have a gel electrophoresis pattern showing four bands.
Add this pattern to your gel in well 3 in number 27.b. and explain below why the gel shows a four-band pattern.
[2]
29. a.
What is the purpose of a Southern blot?
b. What two techniques discussed earlier in this chapter are used in performing a Southern blot? [2]
c.
Label the diagram outlining the Southern Blotting of DNA Fragments.
30. In your own words, what is ‘DNA sequencing’ (do not give me the uses of sequencing data obtained
through DNA sequencing, but explain to me what this sequencing data is that you get from performing
this technique)?
31. We already studied genetic mapping in past chapters. This chapter introduces you to techniques used in
physical mapping. Based on your knowledge of each term, why is genetic mapping considered a “relative
mapping” as opposed to physical mapping? [3]
32. a. What exactly is the difference between a ribonucleotide of RNA, a deoxyribonucleotide of DNA, and
dideoxyribonucleotide used in automated DNA sequencing methods and the Sanger method of DNA
sequencing.
b. Why does a dideoxyribonucleotide terminate a growing DNA strand? You may need to refer to Figure 16.14.
33. a. Explain the application and methodology of the Dideoxy Chan Termination Method of DNA sequencing.
b. Why are the four nucleotides in DNA each labeled with a different color of fluorescent tag?
c. Label the illustration showing the reactants and DNA sequencing procedure described above.
34. Describe the usefulness AND the three-step procedure of RT-PCR?
35. a. What are ‘microarrays’ (“DNA chips”) used for?
b. Label the following illustrating explaining the general technique for using microarrays and how the
results are interpreted.
36. Explain how microarrays are used in understanding gene expression patterns in normal & cancerous tissue. [2]
37. What is ‘in vitro mutagenesis’ and what does it help scientists understand? [3]
Cloning organisms may lead to production of stem cells for research and other applications. [2]
38. Define the following terms.
a.
Organismal cloning:
b. Reproductive cloning:
39. Distinguish between Pluripotent and Totipotent Cells.
40. How is Nuclear Transplantation performed in animals? [2]
41. Use unlabeled Figure 20.18 to explain the six steps in reproductive cloning for animals. [2]
1.
2.
3.
4.
5.
6.
42. a. What is a stem cell?
b. What is an Induced pluripotent stem cell (iPS)?
c.
How might these resolve the debate about using stem cells for medical treatments?
43. What are the differences between cloning a plant versus an animal?
44. Is a cloned animal identical to its parent? In what ways is it and in what ways isn’t it? Why might this be?
45. How are embryonic stem cells (ES) different from adult stem cells (AS)?
The practical applications of DNA technology affect our lives in many ways. [2]
46.
a.
What is a ‘polymorphism’?
b. What are single nucleotide polymorphisms (SNPs).
c.
Define and explain the significance of RFLP’s – restriction length polymorphisms. [2]
d. In question 27, you used the understanding of SNPs and RFLPs. Explain how SNPs and RFLPs were
demonstrated in analyzing sickle-cell alleles. [2]
47. a.
What is ’gene therapy’?
b. What are the basic steps in human gene therapy with a retroviral vector?
c.
Based on your knowledge of genes and the control of gene expression, how may this procedure harm an
individual?
48. Now that the technology is starting to exist, should we remove all harmful alleles from organisms gene
pools? Why or why not?
49. Explain how transgenic “pharm” animals might be able to produce human proteins. [2]
50. What is a ‘genetic profile’ (“DNA fingerprint”)?
51. a. What are ‘short tandem repeats (STRs)’?
b. In what ways are STRs and genetic profiles useful in forensic and paternity cases?
52. What is a ‘transgenic animal’?
53. How are Ti plasmids used in agriculture and genetic engineering in plants?
54. a. What are ‘GMOs’ and why do we want to produce them?
b. What are some of the different properties introduced into plants through genetic engineering? [1]
b. Do you think that you have eaten any genetically modified foods? [2]
55. Please answer the Self-Quiz at the end of your chapter. Do your best to try it from memory first in order to
test how well you grasped the material.
1. ______
2. ______
3. ______
4. ______
5. ______
6. ______
7. ______
8. ______
9.
References
1.
Campbell et al. (2008). AP* Edition Biology. 8th Ed. San Francisco: Pearson Benjamin Cummings.
2.
Adapted from Fred and Theresa Holtzclaw
3.
Adapted from L. Miriello