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Molecular Genetics and Biotechnology Practice Test (page 3)

By — McGraw-Hill Professional
Updated on Aug 23, 2011

Answers

Vocabulary

  1. electrophoresis
  2. denaturation or melting
  3. renaturation or annealing
  4. autoradiography
  5. palindrome
  6. restriction endonucleases
  7. gene (DNA) library or genomic library
  8. terminal (deoxyribonucleotidal) transferase
  9. polymerase chain reaction
  10. complementary DNA (cDNA)

Multiple-Choice

  1. d
  2. a
  3. c
  4. b
  5. e
  6. e
  7. a
  8. d
  9. e
  10. c

Molecular Genetics and Biotechnology

  1.   (a) 51.02   (b) 40.82   (c) 73.47
  2.   (b), because it has a higher (G + C) / (A + T) ratio
  3. Molecular Genetics and Biotechnology Answers to Supplementary Problems
  4. Of all the RNAs that hybridize with DNA, the 16S and 23S rRNAs account for only 0.0014 + 0.0018 = 0.0032 or 0.32%. Since these rRNAs and mRNAs are about equally represented in a cell, the ratio 1/0.0032 = 312.5 expresses how much more active in transcription are the genes for the rRNAs.
  5. Attach anti-rat insulin antibodies to a plastic disk about the size of an agar plate. Impress the disk onto the plate and allow any secreted insulin to be specifically bound by the antibodies. Remove the plastic disk and expose it to radioactive anti-insulin antibodies, forming an "immunological sandwich" with the antigen (insulin) between two antibody molecules. Wash away any unattached radioactive antibodies and then make an autoradiograph of the disk. Images on the film can be used to identify the locations of insulin-secreting clones on the agar plate.
  6. More fragments are expected from HaeIII because the probability of a specific four-base sequence is greater than the probability of a specific six-base sequence if the nucleotides are distributed along a chain in essentially a random order.
  7. Insert the gene for lambda repressor protein immediately adjacent to the lac promoter in a plasmid. With no operator locus between these two genes, thousands of repressor molecules should be made per cell constitutively.
  8. (a) Five kinds of cells: (1) bacteria that do not contain any plasmid DNA, (2) bacteria that took up the plasmid DNA, but do not contain any human DNA, (3) bacteria that contain the plasmid DNA with human DNA spliced in, but not the desired human gene sequence, (4) bacteria that contain plasmid DNA and a portion of the desired human gene, but not all of it, (5) bacteria that contain plasmid DNA and all of the desired human gene.   (b) Approximately 600,000 recombinant phage, determined as follows.
  9. Molecular Genetics and Biotechnology Answers to Supplementary Problems

  10. Organisms that live in hot springs must have heat-stable enzymes. The first thermostable DNA polymerase used in automating PCR was isolated from a bacterium called Thermus aquaticus that normally lives in hot springs at a temperature of 70-80°C. Consequently, its polymerase is stable at temperatures near this point.
  11. (a) The amino acid sequence can be used to construct a degenerateDNAprobe (see Example 12.5) that can be labeled with radioactivity.   (b) A cDNA library is most appropriate since this is a mammalian gene and it may contain introns. Agenomic library will most likely allow the isolation of gene fragments, many of which may contain the introns. An expression library is more difficult to make, and useful if an antibody "probe" is being used.
  12.  
    1. Steps in cDNAproduction: (1) isolateRNAfrom cells, (2) add reverse transcriptase enzyme, dNTPs and oligo-dT primers to make a first strand of cDNA, (3) degrade RNA, (4) add DNA polymerase I to make a second strand of cDNA, (5) clip the hairpin using S1 nuclease.
    2. Since the gene for protein P is 1000 bp or less in length, it is too small to clone into phage lambda, which requires fragments of approximately 15,000 bp. Thus, we should choose plasmid pBR322 and clone DNA fragments ranging from 2000 to 3000 bp. This will ensure that the entire gene will be found in one fragment, as well as its associated regulatory sequences.
    3. Cut the cDNAs and the pBR322 vector with PstI enzyme, then ligate the cDNAs with the cut plasmid.
    4. By employing the replica plating technique, we can select cells that are ampicillin-sensitive and tetracyclineresistant. Only those cells that have incorporated the plasmid are tetracycline-resistant, and of these only the cells with an insert in the ampR gene are ampicillin-sensitive.
    5. Blot each Petri plate with a nitrocellulose filter paper, thereby transferring some cells of each clone onto the filter for in situ hybridization. Lyse the cells and denature the DNA with a dilute sodium hydroxide solution. Single strands of the denaturedDNA thus will stick to the filter. Flood the filter with the radioactive probe. After washing to remove any of the unhybridized probe, subject the filter to autoradiography. Select cells from the plate that correspond in position to the radioactive loci on the filter.
    6. Cut the plasmids with the PstI enzyme. This should liberate the gene fragment, assuming that there are no PstI sites within the gene fragment.
    7. The gene for protein P should hybridize specifically with the probe on a Southern blot. Also, by sequencing the gene fragment, it will be possible to ascertain the genetic code and then compare this to the original amino acid sequence obtained and any other previously cloned relatives of protein P using bioinformatics. If protein P has a measurable activity (e.g., enzymatic conversion of clear substrate into a color), the protein might be synthesized in an in vitro translation system and its activity checked. Finally, the gene might be cloned into an expression vector and the protein produced in E. coli (or another appropriate system). This protein could also be tested for activity
  13. After digestion with EcoRI, the normal gene for protein P should produce one band, 700 bp long. The mutant gene for an abnormal protein P should produce one band of at least 850 bp due to the loss of one of the EcoRI sites. The size of the band is dependent on where the next EcoRI site may reside in the neighboring flanking genomic DNA.
  14. Note: The migration distance is not linear with increasing fragment length, but more like a log function.
  15. Molecular Genetics and Biotechnology Answers to Supplementary Problems

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