BITC1311 Intro to Biotechnology Name
Chapter 3. History of Genetic Manipulation: Recombinant DNA Technology
Thieman & Palladino Guided Notes
Using your textbook and its glossary, define the following terms: (A “genetics glossary” at http://helios.bto.ed.ac.uk/bto/glassary/ and a “biotechnology glossary” at www.biotechterms.org
may also be useful.)
Recombinant DNA technology
Genetic engineering
Model organisms
Gene cloning
Vectors
Transformation
Electroporation
Antibiotic selection
Replica plating
2.
Genetic transformation of bacteria had been discovered in the 1920’s, but gene cloning became possible only after restriction enzymes and plasmids became available in the 1970’s. a.
Where do restriction enzymes come from, and what are their biological roles? b.
What is the difference between an endonuclease and an exonuclease? c.
Where do restriction enzymes cut DNA molecules? d.
What is a palindrome, and what does this have to do with restriction enzymes? e.
How do restriction enzymes produce “sticky ends” (aka “cohesive ends” or “overhangs”)? f.
What is the advantages to using a restriction enzyme that makes sticky ends or blunt ends?
3.
In what ways do plasmids make convenient vectors? a.
How have plasmids been genetically engineered in order to improve their usefulness? (p. 60) b.
What advantages and limitations do the following vectors have?
Bacteriophage
Cosmids
BACs
YACs
Ti plasmid
c. How must a vector be engineered in order for it to be an expression vector?
4.
Many processes in molecular biology use a “hybridization” technique a.
What must be done to make a hybridization probe detectable? b.
A DNA probe must be able to hybridize to DNA by complementary base-pairing. What must be done to the DNA being probed in order for it to be accessible to the probe? c.
How are oligonucleotide probes constructed?
5.
When Bubba discovered that his cousin Jack was taking the ACC Introduction to Biotechnology course, he sent a letter asking Jack to clone the growth hormone for crayfish in order to produce giant crayfish in his bayou.
Write a description of all the steps that Jack must take in order to successfully clone crayfish growth hormone into a bacterium for Bubba. Pay attention to the following considerations: a.
all the materials and enzymes required during this process b.
specifics about the selectable marker gene used in the plasmid c.
source and processing of nucleic acids for the library d.
details about how the library could be screened for transformants e.
details about how the growth hormone gene might be screened for f.
details about how the growth hormone gene might be expressed at high levels g.
whether PCR could be used in this process
6.
What does PCR stand for? a.
Why is a primer necessary for PCR amplifications of DNA? b.
Why are two primers necessary? c.
What unique type of DNA polymerase is used in PCR amplifications of DNA? What is the advantage of this type of DNA polymerase, compared to the DNA polymerase that can be isolated from other prokaryotes or eukaryotes? d.
During the thermocycling of a PCR run:
1.
What happens at the 94-96 o temperature setting? Why is this step necessary?
2.
What happens at the 50-65 o temperature setting? Why is this step necessary?
3.
What happens at the 72 o temperature setting? Why is this step necessary?

f.
Why are multiple cycles of PCR thermocycle necessary? How many copies of each original chromosomal
DNA template are there made after 10 thermocycles? After 20 thermocycles? After 30 thermocycles?
7.
How many countries participated in the HGP? a.
When was the rough draft of the HGP sequence of the human genome completed? b.
How much of the human genome was covered by the rough draft? c.
In comparitive genomics, DNA sequences and genes are compared between species. How many genes do we share with yeast? With mice?
8.
What are the forces that separate DNA fragments in agarose gel electrophoresis? a.
Do the smaller fragments of DNA migrate faster or slower though the agarose gel during electrophoresis?
Why? b.
How does one change the conditions of electrophoresis when the DNA fragments are especially small? c.
How does one speed up the separation by electrophoresis? d.
What limits the upper range of speed of electrophoresis? e.
DNA is invisible. How are the DNA fragments separated by electrophoresis made visible?
9.
How is a restriction map made for a given piece of DNA? Why is a restriction map useful?
10.
What are the reactants, enzymes, and cofactors required for DNA sequencing ? a.
What causes the DNA fragments to vary in size? b.
How are the DNA fragments detected in the electrophoretic gel
11.
What is the FISH technique used for?
12.
How are the DNA fragments prepared prior to the blotting step of a Southern blotting experiment?
b. How are the DNA fragments separated from each other? c. Why are the separated fragments not resolved from each other, forming a “smear” following this separation? d.
What exactly are these “smears” of DNA fragments blotted to? Why is this step necessary? e.
How are the DNA “probes” made for the hybridization step? f.
What is the purpose of these DNA probes?
13.
What is the difference between a Southern blot and a Northern blot? a.
What is the purpose of a Northern blot? b.
What advantages are there to using RT-PCR for expression analysis? What special enzyme is used in this technique? c.
In Figure 3.16 (b), what is the reasoning behind the “negative controls” in lanes 2 & 3? What is the reasoning behind the amplification of actin mRNA in lanes 4, 6, 7, and 8? d.
What advantage is there to using in situ hybridization for expression analysis?
14. Go to www.geneticsplace.com
and select the book by Campbell & Heyer : Discovering Genomics, Proteomics, and Bioinformatics . Select the “ DNA microarray ” method in Chapter 4 and answer the following questions: a.
Describe the steps used to perform a microarray experiment for comparing gene expression of yeast grown under aerobic and anaerobic conditions. b.
Approximately how many genes are there in the yeast genome? How are these genes accounted for in the
DNA microarray? c.
Click on the picture of the resulting DNA microarray. Visually estimate how much overlap is there in gene expression in yeast grown under the two conditions. d.
In what ways are DNA microarrays similar to and different from a Southern blot?
15.
Bioinformatics is a new and interdisciplinary field. What 3 fields merge in bioinformatics?
16.
Imagine yourself cloning a gene that you have identified as being expressed only in cancer cells. You next sequence this cloned DNA. Explain how you might use the NCBI BLAST software and the GenBank database to determine what this gene is doing in a cancer cell.