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20. The following gure shows a gel of a total and partial digest of a DNA segment treated with HindII. Endlabeled segments are noted by asterisks. Draw the restriction map of the original segment.
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16. A plasmid that contains an EcoRI site within a gene for ampicillin resistance is cut with EcoRI, and then religated. This plasmid is used to transform E. coli cells, and the plasmid is reisolated from the ampicillin-resistant colonies. The reisolated plasmids from two different colonies are electrophoresed, and the results appear in the following gure.
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21. Several mutants of the DNA segment shown in problem 19 were isolated.They gave the following gel patterns when the total digests were electrophoresed. Asterisks denote the end-labeled segments. Can you determine the nature of the mutations
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How do you account for the two bands in colony 2 17. Most human genes contain one or more introns. Since bacteria cannot excise introns from nuclear messenger RNA (snRNPs are needed), how can bacteria be used to make large quantities of a human protein
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18. How are DNA ngerprints useful in forensic cases Could they be used in paternity exclusion 19. The following segment of DNA is cut four times by the restriction endonuclease EcoRI at the places shown. Diagram the gel banding that would result from electrophoresis of the total and partial digests. Note the end-labeled segments and regions where several segments form bands at the same place on the gel. 100
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22. Restriction maps of a segment of DNA were worked out separately for BamHI and TaqI. Two overlays of the maps are possible. The double-digest gel is shown in the following gure (asterisks denote end labels). Which overlay is correct
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III. Molecular Genetics
13. Genomics, Biotechnology, and Recombinant DNA
The McGraw Hill Companies, 2001
Thirteen
Genomics, Biotechnology, and Recombinant DNA
23. A linear DNA molecule 1,000 bp long gives the following size fragments when treated with these restriction enzymes. Derive a restriction map.
EcoRI: BamHI: EcoRI BamHI: 300 bp, 700 bp 150 bp, 200 bp, 250 bp, 400 bp 50 bp, 100 bp, 200 bp, 250 bp, 400 bp
nates ampicillin resistance. If the plasmid is digested completely with enzyme mixes, the following fragments result:
Mixture EcoRI EcoRI EcoRI EcoRI EcoRI Pst I Bgl II HindIII Sal I Bgl II Pst I Fragment Size (kb) 0.7, 2.3 0.3, 2.7 0.08, 2.92 0.85, 2.15 0.3, 0.7, 2.00
24. A linear DNA molecule cut with EcoRI yields fragments of 3 kb, 4.2 kb, and 5 kb. What are the possible restriction maps 25. You have double-stranded DNA that you radioactively label at the 5 ends. Digestion of this molecule with either EcoRI or BamHI yields the following fragments.The numbers are in kilobases (kb), and an asterisk indicates the fragments that are labeled. EcoRI: 2.8, 4.6, 6.2*, 7.4, 8.0* BamHI: 6.0*, 10.0*, 13.0 If unlabeled DNA is digested with both enzymes simultaneously, the following fragments appear: 1.0, 2.0, 2.8, 3.6, 6.0, 6.2, 7.4. What is the restriction map for the two enzymes 26. A 12 kb DNA molecule cut with EcoRI yields one 12 kb fragment. When the original molecule is cut with BamHI, three fragments of 2 kb, 4.5 kb, and 5.5 kb are produced. When the fragment from EcoRI is treated with BamHI, four fragments of 2 kb, 2.5 kb, 3.0 kb, and 4.5 kb are produced. Draw a restriction map. 27. A plasmid 3 kb in length contains a gene for ampicillin resistance and a gene for tetracycline resistance.The plasmid has a single site for each of the following enzymes: EcoRI, BglII, HindIII, PstI, and SalI. If DNA is cloned into the EcoRI site, resistance to either antibiotic is not affected. DNA cloned into the BglII, HindIII, or SalI sites abolishes tetracycline resistance, and DNA inserted into the PstI site elimi-
Draw a restriction map of the plasmid, and indicate the locations of the resistance genes and the sites of enzymatic cleavage. 28. A gene has the following EcoRI restriction map (in kilobases): 1.0 Z 0.7 Z 2.0
Draw the gel pattern expected from a. a mutant that has lost the site between the 1.0 and 0.7 kb fragments. b. a mutant that has a new site within the 2.0 kb fragment. 29. A DNA fragment 8 kb in size is labeled with 32P at the 5 ends. It is then digested with EcoRI, BglII, or a mixture of both enzymes. The size of the fragments and the labeled fragments (*) appear as follows. Sizes are in kilobases.
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