Answers To Review Questions

1. E. All these statements are true. Plasmids used as cloning vectors are circular, have an origin of replication so that they can replicate in the host bacterium, carry antibiotic genes so that transformed cells can be selected for, and have unique restriction enzyme cutting sites to allow genes of interest to be inserted into the plasmid.

2. D. cDNA means DNA that is complementary to the mRNA found in a tissue sample. cDNAs are generated using a poly-T primer that binds to processed mRNA, but which will not bind to tRNA or rRNA, which have no poly-A sequence.

3. A. Addition of the gratuitous inducer lac operon inducer IPTG should ensure good production of the protein, which can then be recognized by the antibody. Considering the other answers: (B) The multiple cloning site makes insertion of the gene of interest easier, but has no implications regarding the method to be used to then screen the library. (C) If one is using an antibody to recognize protein, the presence of DNA is largely irrelevant. (D) The antibody is used to recognize the particular protein coded by the gene of interest. It would therefore be highly counterproductive to destroy the proteins with a protease. (E) There is no reason why one should want to use an antibody that recognizes IPTG.

4. E. The primers bind to the template, allowing DNA polymerase to generate the probe using the deoxynucleotides provided. Because one of the deoxynucleotides carries a radioactive tag, the resulting probe is radioactive and can therefore be used, for example, to reveal the position of complementary DNA on a Southern blot.

5. E. The primer binds to the DNA to be sequenced. DNA polymerase then begins work using the deoxynucleotides, but each time it instead incorporates a dideoxynucleotide; synthesis of that strand stops so that the DNA of that specific length is flagged with a fluorophore of the color corresponding to the base encountered.

6. D. Southern blotting is used to analyze DNA, northern blotting is used to analyze RNA, and western blotting is used to analyze protein (Table 7.2). There is no eastern blotting (although there is a technique called SouthWestern blotting!).

7. C. PCR proceeds by cycles of denaturation (separation of the DNA strands), annealing (of primers onto the strands), and polymerization to form double helices.

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