Download Unit 12 Handout - Chavis Biology

UNIT 12: Biotechnology
Name: ________________________
Essential Idea(s):
Gel electrophoresis is used for DNA profiling.
Biologists have developed techniques for artificial manipulation of DNA, cells and organisms.
IB Assessment Statements
Unit Objectives. Students will be able to:
3.5.U1
Gel electrophoresis is used to separate
proteins or fragments of DNA according to size.

3.5.U2
PCR can be used to amplify small amounts of DNA.
Compare the role of restriction enzymes
in nature and in biotechnology
applications.
2.7.A1
Use of Taq DNA polymerase to produce multiple
copies of DNA rapidly by the polymerase chain
reaction (PCR)

Describe the functioning of restriction
enzymes.
DNA profiling involves comparison of DNA.

Contrast sticky vs. blunt ends.
3.5.U3
3.5.S2
Analysis of examples of DNA profiles.

Explain the function and purpose of DNA
electrophoresis.
3.5.A1
Use of DNA profiling in paternity and forensic
investigations.

Outline the functions of the buffer, marker
and loading dye in DNA electrophoresis.
3.5.U4
Genetic modification is carried out by gene
transfer between species.

Describe the process of DNA profiling.
3.5.A2
Gene transfer to bacteria with plasmids using
restriction endonucleases and DNA ligase.

Read a DNA profile to determine
relatedness or forensic guilt.
3.5.U5
Clones are groups of genetically identical
organisms, derived from a single original parent cell.

Describe the process of DNA
fingerprinting.
3.5.U7
Animals can be cloned at the embryo into more
than one group of cells.

Outline how genetic modification is carried
out.

Evaluate the risks and benefits associated
with Genetically Modified crops.
3.5.U8
Methods have been developed for cloning adult
animals using differentiated cells.
3.5.A4
Production of cloned embryos by somatic-cell
nuclear transfer.
3.5.U6
Many plant species and some animal species have
natural methods of cloning.
3.5.S3
Design of an experiment to assess one factor
affecting the rooting of stem-cutting.
3.5.NOS Assessing risks associated with scientific
research: scientists attempt to assess the risks
associated with genetically modified crops or
livestock.
3.5.A3
Assessment of the potential risks and benefits
associated with genetic modification of crops.
3.5.S3
Analysis of data on risks to monarch butterflies of Bt
crops.
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Restriction Enzymes
WHY ARE RESTRICTION ENZYMES NECESSARY?
1. Restriction enzymes are “molecular scissors” that ___________________________________________
2. Restriction enzymes are isolated from __________________ for use in biotechnology research.
a. The function of restriction enzymes in bacterial cells is to cut
apart foreign DNA molecules (i.e. from
____________________________________)
b. Restriction enzymes are named from the bacterium from
which it was discovered. For example: __________________
i. ______ – the first letter of the ___________ name of the
bacteria (Escherichia)
ii. ______ – the first two letters of the ____________ name of
the bacteria (coli)
iii. ______ – a particular _________________ of this bacteria
(strain RY13)
iv. ______ – the particular ________________ among several produced by this strain
3.
Restriction enzymes cut DNA in areas of specific base pair sequences, called ____________________
a.
In general, a restriction site is a 4- or 6-base-pair sequence that is a ______________________
i. A sequence in which the “top” strand read from 5’ to 3’ is the same as the bottom strand read from 5’ to
3’.
ii. Each different restriction enzyme
(and there are hundreds) has its
own restriction site. The ends of
the DNA produced after being cut
with a restriction enzyme can be
Restriction
Enzyme
EcoRI
BamHI
either “blunt” or “sticky”
4.
When scientists study a DNA molecule, one of the
HindIII
first things they do is figure out where many
restriction sites are. They then create a
AluI
“__________________”, showing the locations of cleavage
sites for many different enzymes.
5.
SmaI
After the restriction enzyme cuts the DNA, the
fragments are of different lengths and can be
HhalI
Restriction site
5’ GAATTC 3’
Sticky or
Blunt
3’ CTTAAG 3’
5’ GGATCC 3’
3’ CCTAGG 5’
5’ AAGCTT 3’
3’ TTCGAA 5’
5’ AGCT 3’
3’ TCGA 5’
5’ CCCGGG 3’
3’ GGGCCC 5’
5’ GCGC 3’
3’ CGCG 5’
separated via ____________________________________
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Restriction Enzyme
Exercises and Questions
Exercise 1: Modeling Restriction Enzyme Action
1. Cut the DNA sequence strips (on the separate half sheet) along their borders. These strips represent double
stranded DNA molecules. Each strip is labeled 1, 2, 3 or 4 in the upper left hand corner.
2. You will now simulate the activity of EcoRI. Scan along the DNA sequence of strip 1 until you find the EcoRI
restriction site. You’ll have to look at your notes to see where on the DNA EcoRI cutes. Using scissors make a
cut through the DNA to simulate the action of the EcoRI restriction enzyme. Separate the two pieces of DNA.
Look at the new DNA ends produced by EcoRI. Are they sticky or blunt? Write EcoRI on the cut ends. Keep the
cut fragments on your desk.
3. Repeat the procedure with strip 2, this time simulating the activity of SmaI. Are the new ends sticky or blunt?
Label the new ends SmaI, and keep the DNA fragments on your desk.
4. Simulate the activity of HindIII with strip 3. Are these ends sticky or blunt? Label the new ends HindIII, and
keep the fragments.
5. Repeat the procedure once more with strip 4 again simulating EcoRI. Pick up the 'front-end' DNA fragment
from strip 4 (an EcoRI fragment) and the “back end” HindIII fragment from strip 3. Both fragments have single
stranded tails of 4 bases. Are the base sequences of the HindIII and EcoRI tails complementary?
6. Put down the HindIII fragment, and pick up the back end DNA fragment from strip 1 (cut with EcoRI). Compare
the single-stranded tails of the EcoRI fragment from strip 1 and the EcoRI fragment from strip 4. Are they
complementary?
7. Imagine that you have cut a completely unknown DNA fragment with EcoRI. Do you think that the single
stranded tails of these fragments would be complementary to the single stranded tails of the fragments
from strip 1 and strip 4? Why or why not?
8. Tape or staple your restriction enzyme fragments for each DNA strand (labeled 1-4) onto the bottom of this
sheet.
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Exercise 2: Identifying Restriction Sites
9. Locate and label the restriction enzyme sites for EcoRI, HindIII, BamHI, and SmaI in the following sequence of
DNA
Exercise 3: Reading Restriction Maps
10. The following is a restriction map of a 4.8 kb (kilobases, means “thousand bases long”) piece of DNA. The first
bases in the strand would be base #1 and the last would be base 4800. The map below shows that there is a
restriction site for HindIII at base # 1400 (1.4kb), a restriction site for XbaI at base #3300 (3.3kb) and a
restriction site for HpaII at base #4200 (4.2kb). What will be the lengths of the fragments produced by the
following restriction enzymes?
a. XbaI only
b. HindIII only
c. HindIII and HpaII
d. HindIII and XbaI
11. A plasmid is a tiny piece of DNA that is separate from the
main circular chromosome of a bacterium. Refer to the
restriction map of the circular plasmid YIP5. This
plasmid contains 5,541 base pairs. There is an EcoRI
restriction site at base pair 1. The locations of other
restriction sites are shown on the map. The numbers
after the enzyme names tell at which base pair that
enzyme cuts the DNA. If you exposed the Y1P5 plasmid
to EcoRI, the restriction enzyme will cut the DNA,
resulting in a linear piece of DNA that is 5,541 base pairs
long.
a. How many DNA fragments would be produced if
the plasmid was exposed to the two enzymes
EcoRI and EagI? How many base pairs long
would each segment be?
b. How many DNA segments would be produced if you took the products from question 11a and exposed
them with PvuII? How many base pairs long would each segment be?
c. How many DNA fragments would be produced if the original plasmid was exposed to the two enzymes
HindIII and ApaI? How many base pairs long would each segment be?
d. How many DNA fragments would be produced if the plasmid was exposed to the three enzymes HindIII,
ApaI, and PvuI? How many base pairs long would each segment be?
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Electrophoresis and the Basics behind DNA Fingerprinting
1. Gel Electrophoresis is the most widely used technique to separate pieces of DNA according to their size
a. View the animation at
http://www.sumanasinc.com/webcontent/animations/content/gelelectrophoresis.html and answer
the following questions:
i. What is the purpose of the restriction enzymes? If the enzymes were not added, how many
bands of DNA would appear on the electrophoresis gel?
ii. What is the purpose of the agarose
gel?
iii. What is the purpose of adding blue
“tracking” dye to the DNA samples?
iv. Explain why DNA has an overall
negative charge.
v. Why is the fact that DNA has a
negative charge so important in the
gel electrophoresis process?
vi. Explain how an agarose gel can separate DNA fragments of different lengths, including a
description of which size molecules move furthest in the gel.
vii. In one or two sentences, summarize the technique of gel electrophoresis.
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2. Applications of gel electrophoresis:
a. Human DNA can be analyzed to:
i. _________________________________________
ii. _________________________________________
iii. _________________________________________
b. Samples can be obtained from _____________________________________________, including:
i. _________________________________________
ii. _________________________________________
iii. _________________________________________
iv. _________________________________________
c. DNA profiling techniques are also used by scientists in other fields of biology, for example:
i. Conservation biologists use DNA profiling to: _____________________________________
ii. Zoos use DNA profiling to: ______________________________________________
iii. Animal behaviorists use DNA profiling to: _____________________________________
iv. Taxonomists use DNA profiling to: ____________________________________________
v. Evolutionary biologists use DNA profiling to: ____________________________________
DNA Fingerprinting Cases
A Paternity Case
Mr. I.M. Megabucks, the wealthiest man in the world, recently died. Since his death, three women have come
forward. Each woman claims to have a child by Megabucks and demands a substantial share of his estate for
her child. Lawyers for the estate have insisted on DNA typing of each of the alleged heirs. Fortunately,
Megabucks anticipated trouble like this before he died, and he arranged to have a sample of his DNA for
analysis. Laboratory technicians used RFLP analysis to look at three highly variable chromosome regions. The
results are shown below. Your job is to analyze the data and determine whether any of the children could be
Megabucks’ heir. Could any of them be
Megabuck’s children? Why or why not?
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The Case of the Bloody Knife
Late one April night, government agents received an
anonymous tip that the Smithsonian Museum was
about to be robbed of its priceless jewel collection.
When the FBI agents arrived at the museum, they
saw that they were too late: the jewels were gone.
Lying facedown on the floor next to the empty
jewel case was the body of a man. The chief
inspector recognized the man as Heinrich Milhouse.
Milhouse had been shot in the chest at close range;
his clothes were saturated with blood. Underneath
the body, the inspectors found a bloody knife. At
the Washington International Airport the next day,
police apprehended Englewood Smink, the
murdered thief’s occasional
partner in crime. Smink denied all knowledge of the murder and the theft. When asked about the fresh cut on his hand, Smink
said that he had had an accident in the kitchen that morning. Suspicious, the chief FBI inspector ordered DNA tests on the vi
ctim, the blood on the victim’s clothes, the blood on the end of the knife found under the victim, and Smink. The results of
the test are shown in the figure above. Should Smink be released? Why or why not?
Genetic Relatedness of Bears
According to the DNA gel results, which of the five
species listed are most closely related?
How do you know? Which one is the most
genetically distinct? How do you know?
Restriction Enzyme Analysis
Below is a plasmid with restriction sites for BamHI and EcoRI. Several restriction digests were performed using these two enzymes
either alone or in combination.
A. Which lane shows a digest with Bam HI only?
B. Which lane shows a digest with EcoRI only?
C. Which lane shows the fragments produced when the plasmid was incubated with both EcoRI and BamHI?
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Polymerase Chain Reaction (PCR)
DNA Amplification
 Using the technique called ________________ ___________ ___________ (PCR), researches are
able to create vast quantities of DNA identical to trace samples.
o Also known as __________ ________________.
 Many procedures in DNA technology require substantial amounts of DNA to work with, for example”
o
o
o
o
o
 Samples from sources may be difficult to obtain in any quantity. Examples of source samples:
o
o
o
PCR Equipment
 Amplification of DNA can be carried out with simple-to-use ___________ _____________ called
________________ ______________.
o Commonly found in the biology departments of ____________ and other kinds of __________
and analytical ________________.
 Amplifies DNA
1.
2.
Primer annealed
3.
4.
5.
+
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PCR Cycles
Cycles of DNA can replicate DNA at an ______________ rate and can make billions of copies of DNA in only a
few hours. An example is shown below (draw it).
PCR
Cycle
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
No. of Target
DNA strands
2
16
128
256
1024
4096
32 768
65 536
262 144
2 097 152
4 194 304
16 777 216
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Gene Technology
What is Gene Technology?
 Gene technology is a broad field which includes analysis of DNA as well as ____________________
and other forms of genetic modification.

Genetic engineering refers the artificial manipulation of genes: adding or subtracting genes, or
changing the way genes work.

Organisms with artificially altered DNA are referred to as _____________________________ (GMOs).

Gene technologies have great potential to benefit humanity through:
o increasing ____________________
o reducing ______________________
o
increasing ____________________
o
producing _____________________
o
preventing and _________________
o
detecting and __________________
Why gene technology?
 Despite potential benefits, gene technology is highly controversial.

Some people feel very strongly that safety concerns associated with the technology have not been
adequately addressed.
BENEFITS
Environmentally friendly
Could improve the
sustainability of crop and
livestock production
Could potentially benefit
the health of many
More predictable and
directed than
selective breeding
RISKS
Who owns and regulates
the GMOs?
Third world economies are
at risk of exploitation
Biological risks have not
been adequately addressed
Animal ethics issues
The costs of errors

One of the most notable accomplishments of genetic engineering is the production of human ________
from bacteria, replacing the often troublesome and limited source from cattle and pigs.

Genetically modified crop plants could ________________________________ or allow utilization of
marginal lands such as those with high salt content.

Inserting normal genes into the bodies’ cells of an organism to correct a genetic defect is called
____________________

More controversial is _______________________, the insertion of genes into a normal individual to
influence a particular trait (“designer babies”)
GMOs may be created by modifying their DNA in one of three ways:
o
___________________________: A foreign gene is added which will enable the GMO to carry out
a new genetic program. Organisms altered in this way are referred to as _______________.
Host DNA
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o
___________________________: An existing gene already present in the organism may be
altered to make it express at a higher level (e.g. growth hormone) or in a different way (in tissue that
would not normally express it). This method is also used for _______________.
Existing gene
altered
Host DNA
o
___________________________: An existing gene may be deleted or deactivated to prevent the
expression of a trait (e.g. the deactivation of the ripening gene in tomatoes).
Host DNA
Steps in Adding a Gene
1. Use a __________________________ to cut out the gene of interest from it’s ____________________
2. Use _______________ restriction enzyme to cut open the DNA strand of the organism being given the
gene. Must be the same enzyme so the “_____________________” are complementary
3. When the two matching “sticky ends” come together, they join by base pairing. This process is called
_______________________. This can allow DNA fragments from a different source, perhaps a
plasmid, to be joined to the DNA fragment. The joined fragments will usually form either a linear
molecule or a circular one.
4. DNA fragments produced using restriction enzymes may be reassembled by a process called
________________. Pieces of DNA are joined together using the enzyme ____________________.
Two pieces of DNA
are cut using the
same restriction
enzyme.
Plasmid
DNA
fragment
This other end of the
foreign DNA is
attracted to the
remaining sticky end
of the plasmid.
The two different DNA
fragments are attracted to each
other by weak hydrogen bonds.
AATT
C
G
G Foreign DNA fragment
CTTA
A
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Ethics of Bioengineering
EXAMPLE
Why was the
organisms
modified?
Technique /
procedure for the
modification
Potential Benefits
of the
modification
Potential risks of
the modification
BT Corn
Golden Rice
Antithrombin
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Cloning
Click the following link and answer the questions that
follow: https://www.genome.gov/25020028.
1. In your own words, define cloning.
2. Explain how clones occur naturally.
Give an example.
3. What is artificial cloning? How many types are there?
4. What is the most common type of cloning that takes place at NHGRI?
5. Briefly explain how genes / animals are cloned.
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6. Give examples of animals that have been cloned. Do they always look identical? Why or why not?
7. Have humans ever been cloned? Why or why not?
8. Discuss the ethics behind animal cloning.
9. What is therapeutic cloning and what are some applications of it? The drawbacks?
10. Considering the ethics of cloning, what are your overall thoughts on this process?
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