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Tamarin: Principles of Genetics, Seventh Edition
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App. A: Brief Ans. to Selected Exercises, Problems, and Critical Thinking Ques.
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Appendix A Brief Answers to Selected Exercises, Problems, and Critical Thinking Questions
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9. Chromosome walking is a technique for cloning overlapping chromosomal regions starting from an arbitrary point (see g. 13.38). It is useful for determining relative locations of genes in uncharted regions as well as cloning regions too big to t in a single vector. 11. Southern and northern blotting are gel transfer techniques used to probe for DNA and RNA sequences, respectively. Western blotting is a technique used for locating a protein by antibody recognition. Dot blotting is a probing technique for cloned DNA that eliminates the electrophoretic separation step. 13. Plasmids of E. coli origin survive in yeast when a yeast centromere (CEN region) is added, allowing them to replicate within the yeast cell. Inactivated SV40 viruses can function in the presence of intact helper viruses that allow them to complete their life cycles. Phage has parts of its chromosome that can be removed while still allowing it to complete its life cycle. 15. Partial digestion of molecule 2 leads to the following molecule: AAAAAAAA TTTTTTTT Some of these molecules will form a circle with the single-stranded Ts paired with single-stranded As. The circle eliminates the free 5 phosphate, and the enzyme can no longer work. 17. We must insert DNA that has no introns into bacterial plasmids. This DNA can be obtained by isolating mature, cytoplasmic messenger RNA and then using reverse transcriptase to make doublestranded cDNA. Plasmids with cDNA inserted can then be used to produce human proteins (expression vectors). 19. Electrophoretic bands of the total digest are (* indicates end label) 50, 100*, 150*, 250, 300 bp. Bands of the partial digest are 50, 100*, 150*, 250, 300( 2), 350, 400*( 2), 450*( 2), 600, 700*, 750*, 850* bp.
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21. Mutant A: elimination of site between the 300- and 50-bp segments. Mutant B: elimination of site between 100- and 300-bp segments. Mutant C: creation of a new site within the 300-bp segment, dividing it into 75- and 225-bp segments. 23. This problem begins as a trial and error attempt to overlay two restriction maps, made more dif cult by the fact that one enzyme, BamHI, has made three cuts that are unordered, leaving many possibilities. However, a bit of thought beforehand makes this problem much easier. If you compare the double digest with the BamHI digest, they share 200-, 250-, and 400-bp segments. The double digest has 50- and 100-bp segments replacing the 150-bp segment in the BamHI digest.The inference is that there is an EcoRI cut in the 150bp segment leading to the 50- and 100-bp segments, with all other segments of the BamHI digestion left uncut. That leaves only two possibilities, as shown below; the data are insuf cient to distinguish between the two choices. EcoRI 300 Z 200 Y 150 Y BamHI BamHI 25. EcoRI BamHI 6.2 700 [any order] EcoRI 300 Z 250 Y 150 Y BamHI BamHI 8.0 6.0 700 [any order]
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We know that the 6.2 and 8.0 kb EcoRI fragments are at opposite ends, and that the 10.0 and 6.0 kb BamHI fragments are at the ends. Therefore, the BamHI 13.0 kb fragment must be in the middle. If the 6.2 and 6.0 kb fragments are at the same end, a double digest should produce a fragment of 0.2 kb. This is not seen, so they are at opposite ends: 6.2 10.0 (2.8, 4.6, 7.4) 13.0 8.0 6.0
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If 7.4 is next to 6.2, we should see a 3.8 fragment. Similarly, if 4.6 is next to 6.2, we should see a 3.8 fragment. We see neither of these fragments, so 2.8 is next to 6.2. The 4.6 fragment must be next. 27. Since EcoRI does not eliminate either resistance, its site must be between the tet r and ampr genes. The PstI site must be within the ampr gene, since insertion of DNA into this site eliminates ampicillin resistance. By similar logic, the other sites must be in the tet r gene. In the double digests, the smaller fragment must represent the distance from the Eco RI site to the other site. We can draw part of the plasmid as:
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