Clinical genetics assignment
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Module10CHomework.pptxHomework 10C This is worth 20 points
Clinical Genetics
Why is the DNA of a microorganism not degraded by a restriction endonuclease it produces, even when it has the restriction sites? (1 pt)
Your job is to clone a gene into a Bluescript plasmid. This is a restriction map of the area with the gene. Describe how you would do it, including the enzymes, the media and the host cells, and how you know which colonies have the gene. A map of Bluescript is on the next slide. 5 points, because there should be 5 points in the answer.
Your favorite gene
XbaI PstI EcoRI
EcoRI
HindIII
HindIII
SalI
Bluescript
This is a restriction map of a segment of DNA. Eco and PST refer to locations where EcoRI and PstI cut the DNA. The restriction sites are numbered as well. The distances between the sites are shown on the line in bp. The thick line represents the part of the molecule that is complementary to the probe.
There will be 5 questions about patients regarding this molecule. You need to know this: If the probe matches anything in the segment, the entire size of the segment will be found. For example, the 4000 bp fragment in the center will produce a 4000 bp size on a Southern Blot even though the probe doesn’t cover the entire sequence.
Pst
2
Eco
5
Pst
4
Eco
1
Pst
6
Eco
3
5000 bp
3000 bp
5000 bp
4000 bp
2000 bp
Individual 1 has restriction sites 1 through 6. If DNA is digested with PstI, what are the expected sizes of the DNA fragments that will hybridize with the probe? (1 pt)
Pst
2
Eco
5
Pst
4
Eco
1
Pst
6
Eco
3
5000 bp
3000 bp
5000 bp
4000 bp
2000 bp
Individual 2 has a mutation that eliminates site 4. If DNA is digested with PstI, what are the expected sizes of the DNA fragments that will hybridize with the probe? (1 pt)
Pst
2
Eco
5
Pst
4
Eco
1
Pst
6
Eco
3
5000 bp
3000 bp
5000 bp
4000 bp
2000 bp
Individual 3 has a mutation that eliminates site 5. If DNA is digested with PstI, what are the sizes of the fragments that will hybridize with the probe? (1 pt)
Pst
2
Eco
5
Pst
4
Eco
1
Pst
6
Eco
3
5000 bp
3000 bp
5000 bp
4000 bp
2000 bp
If the DNA of individual I is digested with both PstI and EcoRI, what are the expected sizes of the DNA fragments that will hybridize with the probe? (1 pt)
Pst
2
Eco
5
Pst
4
Eco
1
Pst
6
Eco
3
5000 bp
3000 bp
5000 bp
4000 bp
2000 bp
If the DNA of individual 3 is digested with both PstI and EcoRI, what are the expected sizes of the DNA fragments that will hybridize with the probe? (1 pt)
Pst
2
Eco
5
Pst
4
Eco
1
Pst
6
Eco
3
5000 bp
3000 bp
5000 bp
4000 bp
2000 bp
You have isolated a mutant of Shigella dysenteriae bacteria that is resistant to kanamycin, and you want to find out what gene is responsible. Design a protocol using genetic selection to identify it. 4 pts
One of the procedures for cloning foreign DNA segments takes advantage of restriction endonucleases such as HindIII that produce complementary single strand ends (sticky ends). These enzymes produce identical sticky ends on cleaved foreign DNA and on the vector DNA into which you insert your DNA. Assume you have inserted your favorite gene into the HindIII site of the Bluescript cloning vector with ligase, you amplified it in E.coli, and isolated a large quantity of the plasmid with the insert. How do you excise your favorite gene from the plasmid? (1 pt)
When you purchase a cloning vector, it contains an origin of replication, a selectable marker gene, and one additional component. 1. Name a selectable marker you have experience with from this class. 2. What other component is in the vector and what is its role? (3 pt)
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Match the type of blot to its target (from Module 10A) 1 pt – all or nothing
| 1. | Western blot | _____ RNA |
| 2. | Southern blot | _____ DNA |
| 3. | Northern blot | _____ protein |
| _____ antibody |
