Chapter12.txt

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melancholyofcj
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84181
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Chapter12.txt
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2011-05-06 10:26:46
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Chapter Microbiology Chang
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Chapter 12 Dr.Chang
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  1. What is molecular cloning?
    Isolation and incorporation of a piece of DNA into a vector so it can be replicated and manipulated.
  2. What are the three steps of gene cloning?
    • 1) Isolation and fragmentation of source DNA
    • 2) Inserting DNA fragment into cloning vector
    • 3) Introduction of cloned DNA into host organism
  3. What is the first step of gene cloning?
    Isolation and fragmentation of source DNA
  4. What is the second step of gene cloning?
    Inserting DNA fragment into cloning vector
  5. Most vectors are derived from ____
    Plasmids or viruses
  6. DNA is generally inserted ____
    In vitro
  7. What is the function of DNA lygase?
    Enzyme that joins two DNA molecules together
  8. What is the third step of gene cloning?
    Introduction of cloned DNA into organism
  9. ____ is often used to get recombinant DNA into host
    Transformation
  10. What are plasmids?
    Natural vectors that have useful properties as cloning vectors
  11. What are the properties of plasmids?
    • -Small size; easy to isolate DNA
    • -Independent of origin of replication
    • -Multiple copy number; get multiple copies of cloned gene per cell
    • -Presence of selectable markers
  12. Plasmids are ____ of origin of replication
    Independent
  13. What is Blue/White screening?
    • Blue colonies do not have foreign DNA inserted into vector
    • White colonies have foreign DNA inserted
  14. When plasmids are used as cloning vectors, _____ colonies do not have foreign DNA inserted into vector while ____ colonies had foreign DNA inserted into vector
    Blue ; white
  15. What is insertional activation?
    • lacZ gene is inactivated by insertion of foreign DNA
    • -Inactivated lacZ cannot process Xgal; blue color does not develop
  16. When inactivated lacZ cannot process Xgal, _____ does not develop
    Blue color
  17. What is the chemical process of insertional activation?
    B-galactosidase --> x-gal substrate --> produces blue color
  18. What is an example of a common cloning vector?
    pUC19
  19. What is pUC19 and what does it contain?
    • 1) Common cloning vector
    • 2) Contains ampicillin resistance and lacZ gene
    • 3) Contains polylinker (multiple cloning site) within lacZ gene
  20. What is a polylinker and where is it found?
    Multiple cloning site within lacZ gene ; found in pUC19 cloning vector
  21. What is sequencing?
    Determining the precise order of nucleotides in a DNA or RNA molecule
  22. _____ is determining the precise order of nucleotides in a DNA or RNA molecule
    Sequencing
  23. What is the Sanger dideoxy method and what is it used for?
    • -Uses dideoxy analogs of dNTPs used in conjunction with dNTPs
    • -Analog prevents further extension of DNA chain
    • -Used for sequencing unknown DNA
  24. How do you make the DNA primer for unknown sequences?
    Use random hexomers (random primers)
  25. What are hexomers used for?
    Random DNA primers used for sequencing, using the Sanger method
  26. Why is the 454 sequencing system more advantageous?
    It generates data 100X faster than the Sanger method
  27. The 454 sequencing system relies on two major advances. What are they?
    • -Massively parallel liquid handling
    • -Pyrosequencing
  28. In the 454 sequencing system, _____ is released each time a base is added to DNA strand
    Light
  29. In the 454 sequencing system, light is released when ____
    A base is added to the DNA strand
  30. In pyrosequencing, name the 4 enzymes involved in the reaction
    • 1) DNA Polymerase
    • 2) ATP sulfurylase
    • 3) Luciferase
    • 4) Apyrase
  31. In pyrosequencing, which enzyme converts PPi to ATPi?
    ATP sulfurylase
  32. In pyrosequencing, which enzyme converts ATP to light?
    Luciferase
  33. In pyrosequencing, which enzyme removes the nucleotides?
    Apyrase
  34. In pyroseqencing, what is the function of ATP sulfurylase?
    Converts PPi to ATPi
  35. In pyrosequencing, what is the function of Luciferase?
    Converts ATP to light
  36. In pyrosequencing, what is the function of Apyrase?
    Removes the nucleotides
  37. What is shotgun sequencing?
    Entire genome is cloned and resultant clones are sequenced
  38. The process in which the entire genome is cloned and resultant clones are sequenced is called ____
    Shotgun sequencing
  39. What is the difference between Closed and Draft genomes?
    Closed genome relies on manpower while draft genome does not
  40. What does PCR stand for?
    Polymerase Chain Reaction
  41. What is the Polymerase Chain Reaction (PCR)?
    Method that produces multiple copies of DNA in vitro
  42. Where does the PCR process happen?
    In vitro
  43. The method that produces multiple copies of DNA in vitro is called _____
    Polymerase Chain Reaction (PCR)
  44. What enzyme does PCR use?
    DNA Polymerase
  45. What is the name of the enzyme used for PCR and why is it used?
    Taq Polymerase ; used because it is heat tolerant/thermostable
  46. In what instrument is PCR performed?
    Thermocycler
  47. What are the three steps of PCR and at what temperatures do they occur?
    • 1) Denaturation ; 94C
    • 2) Annealing ; 50-60C
    • 3) Extension ; 72C
  48. What is annotation?
    Converting raw sequence data into a list of genes present in the genome
  49. The process of converting raw sequence data into a list of genes present in the genome is called _____
    Annotation
  50. What is Functional ORF?
    An open reading frame that encodes a protein
  51. An open reading fram that encodes a protein is called ____
    Functional ORF
  52. In Functional ORF, computer algorithms search for what?
    Start/stop codons, Shine-Dalgarno sequences, -35 and -10 regions
  53. What is the thermostable DNA Polymerase?
    Taq polymerase
  54. Taq polymerase is stable at what temperature?
    90C
  55. What is the difference between Taq polymerase and Pfu polymerase?
    Pfu is more thermostable than Taq and Pfu has proofreading activity while Taq does not
  56. ____ polymerase has proofreading activity while ____ polymerase does not
    Pfu ; Taq
  57. During each round of PCR, the amount of product ____
    Doubles
  58. Why is PCR an important technique?
    • 1) Exponential increase in DNA
    • 2) Only few molecules of target DNA are needed
    • 3) PCR is valuable for cloning and sequencing purposes
    • 4) PCR has been used to amplify DNA from mummified remains, fossilized plants and animals
  59. PCR has been used to amplify DNA from _____, _____ and ______
    Mummified remains ; Fossilized plants ; Animals

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