ZOO2144 Practical 2: Gene technology
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Name:
Student number:
Question 1. Enzymes used in Recombinant DNA Technology.
[4]
Answer the following questions:
1.1 What is the function of restriction endonucleases?
(1)
1.2 What is the function of reverse transcriptase?
(1)
1.3 Name one other enzyme that is used in DNA cloning.
(1)
1.4 What is special about Taq DNA polymerase?
(1)
Question 2. Gel electrophoresis and Southern Blotting.
[14]
A researcher isolated DNA from eight different individual organisms that included several
different species. The researcher wanted to compare a certain gene among the eight
organisms, therefore they performed a Southern Blot to visualise the DNA fragments. The
diagram on the next page shows the output from the Southern Blot, which indicates only this
specific gene in each organism. In the diagram, S1 to S8 correspond to the eight genetic
samples, and indicate the position of the wells where the samples were added to the gel. The
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Practical 2 – Principles of Genetics – ZOO2144 – 2023 – University of Venda
column on the left is a series of markers to help analysis of the Southern Blot. The numbers
on the markers do not correspond to fragment size. Answer the questions below the diagram.
2.1 What is the main purpose of gel electrophoresis?
(1)
2.2 To which side of the gel (top or bottom) is the negative pole applied during gel
electrophoresis?
(1)
2.3 DNA has a:
(1)
a) Hydroxyl charge
b) Negative charge
c) Positive charge
d) Kinetic charge
e) Neutral charge
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Practical 2 – Principles of Genetics – ZOO2144 – 2023 – University of Venda
2.4 After electrophoresis is complete, where on the gel will you find the smaller fragments
and where will you find the larger fragments? Explain why: what happens with the fragments
during electrophoresis and how does agarose gel help this process?
(4)
2.5 During the Southern Blotting process, after running the gel, the DNA fragments are
transferred to a filter. How can the gene of interest be made visible on the filter?
(2)
2.6 List the samples that likely came from the same species. Explain your answer(s).
(4)
2.7 Sample S1 has two fragments on the Southern Blot. Assuming that there has been no
cross-contamination of the samples, what do the two fragments imply?
(1)
Question 3. Restriction enzyme mapping
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Data from a digest of a Bacillus anthrax plasmid is provided in the diagram on the next page
so you can do a restriction enzyme map. The Bacillus anthrax plasmid is 5000bp (5Kb) long.
The wells are at the top of the diagram and the left column shows the reference molecular
weight markers. Two restriction enzymes, A and B, were used to obtain two individual digests.
They were combined to produce the third digest. Follow the steps detailed below the diagram
to create the restriction enzyme map.
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Practical 2 – Principles of Genetics – ZOO2144 – 2023 – University of Venda
3.1 Determining the Number of Fragments
(3)
3.1.1 How many fragments were produced by enzyme A? __________
How many cut sites on the plasmid are recognised by enzyme A?_____________
3.1.2 How many fragments were produced by enzyme B? ___________ How many cut sites
on the plasmid are recognised by enzyme B?_____________
3.1.3 How many fragments were produced by the combined digest (A and B)? ___________
Fragment Size
Fragment size is relative to molecular weight and must be determined by comparing the
fragment distance to the molecular weight markers. The fragment sizes have been provided
on the gel pattern for this exercise.
To make a map you must determine the relative positions of the restriction sites on the
plasmid. It helps to know that the double digest is an extension of the two single digests. The
bands you can see in the "A" digest are essentially then subjected to the "B" enzyme digest.
Smaller band sizes seen in the double digest can be combined to add up to the "size" of the
bands in the A digest. In a similar fashion, the smaller bands of the double digest can be
combined to add up to the "size" of the bands in the B digest.
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Practical 2 – Principles of Genetics – ZOO2144 – 2023 – University of Venda
3.2 Starting the Map
3.2.1 Determine which two bands in the double digest make up the smaller A fragment. These
two bands represent where the B restriction site is on the fragment. The line provided below
represents the smaller A fragment. Use this line to mark the approximate position of the B
restriction site and indicate the sizes of the adjacent bands.
(2)
Small A
3.2.2 Determine which two bands in the double digest make up the larger A fragment. These
two bands represent where the B restriction site is on the larger fragment. Mark where the B
restriction site is and indicate the size of each adjacent band.
(2)
Large A
3.2.3 Determine which two bands in the double digest make up the smaller B fragment. These
two bands represent where the A restriction site is on the fragment. Mark where the A
restriction site is and indicate the size of each adjacent band.
(2)
Small B
3.2.4 Determine which two bands in the double digest make up the larger B fragment. These
two bands represent where the A restriction site is on the fragment. Mark where the A
restriction site is and indicate the size of each adjacent band.
(2)
Large B
3.3 Superimposing fragments to map the plasmid.
To determine the relative position of the restriction sites on the plasmid, you need to align
each of the fragments from the single digests to superimpose one on the other and create
one longer fragment.
3.3.1 Align the combined larger A and B fragments on the next page. (Draw the fragments in
the correct positions)
(3)
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Practical 2 – Principles of Genetics – ZOO2144 – 2023 – University of Venda
Large A fragment
Large B fragment
Large A and B superimposed
3.3.2 Do an alignment below for the combined A and B small fragments.
(3)
Small A fragment
Small B fragment
Small A and B superimposed
3.4.1 Align your two superimposed fragments (the superimposed large and the superimposed
small fragments).
(2)
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Practical 2 – Principles of Genetics – ZOO2144 – 2023 – University of Venda
3.4.2 You can now make a linear map of the plasmid by superimposing the aligned fragments
in 3.4.1.
(2)
3.4.3 The data from your linear map can be redrawn to form the circular plasmid with its
restriction sites identified. You can use the 0 mark in the diagram below as your start point.
Indicate the approximate positions of the A and B restriction sites, and the sizes of all
fragments.
(4)
Size
5 Kb
Question 4. First generation DNA sequencing
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The two diagrams on the next page show the gel output for (4.1) the Maxam-Gilbert base
destruction method and (4.2) the Sanger’s chain termination method of sequencing DNA. The
top row indicates the position of the wells and the chemicals that were added to the wells
along with the DNA to be sequenced. Write the nucleotide sequence of each gel output.
Additionally, add the complementary DNA strand to show the double stranded sequence, and
show the 5’ and 3’ ends on both strands.
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Practical 2 – Principles of Genetics – ZOO2144 – 2023 – University of Venda
4.1
4.2
Answers:
4.1
4.2
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Practical 2 – Principles of Genetics – ZOO2144 – 2023 – University of Venda