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MICRB 106 - Exam 3 > Recombinant DNA Technology > Flashcards

Recombinant DNA Technology Flashcards

1
Q

What is biotechnology?

A

The use of microorganisms to make practical products

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2
Q

What is recombinant DNA technology?

A

Genetic engineering

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3
Q

Describe donors and recipients in the context of genetically modified organisms (GMOs) (2)

A
  • Genes are isolated from a donor organism
  • Genes are inserted into the genome of a recipient organism
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4
Q

What was the first drug manufactured using recombinant DNA technology?

A

Insulin

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5
Q

What is reverse transcription?

A

Synthesis of RNA into DNA

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6
Q

Retroviruses carry their genome as ______

A

RNA

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7
Q

Reverse transcriptase is a ______

A

DNA polymerase enzyme

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8
Q

What is the function of reverse transcriptase?

A

Synthesis of cDNA from an mRNA template

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9
Q

How is complementary DNA (cDNA) made?

A

Through reverse transcriptase

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10
Q

How are restriction enzymes produced?

A

Bacteria cut DNA at restriction sites

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11
Q

What characteristics make restriction enzymes ‘restriction endonucleases’? (2)

A
  • Recognizing palindromic sequences
  • Restricting bacteriophages from multiplying
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12
Q

Restriction sites are specific nucleotide sequences called ______

A

Palindromes

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13
Q

Why do bacteria produce restriction enzymes in nature?

A

To stop bacteriophages from multiplying

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14
Q

Describe EcoRI restriction enzymes (2)

A
  • Make staggered cuts of 2 strands of DNA
  • Create sticky ends
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15
Q

Describe Hindlll and Smal restriction enzymes (2)

A
  • Cut both strands of DNA at the same point
  • Create blunt ends
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16
Q

It is more difficult to make recombinant DNA from ______ fragments

A

Blunt-ended

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17
Q

What are vectors in the context of recombinant DNA technology?

A

Nucleic acid molecules

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18
Q

What are some examples of nucleic acid vectors? (2)

A
  • Viral genomes
  • Plasmids
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19
Q

What selectable marker gene do vectors contain?

A

Antibiotic resistance gene

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20
Q

How does the selection for vectors work? (2)

A
  • A medium contains the antibiotic
  • Cells containing the recombinant plasmid can grow on the medium
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21
Q

What is the goal of recombinant DNA?

A

Insert a useful gene into a cell to get a new phenotype

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22
Q

What are ‘CRISPR’s? – what does the acronym stand for?

A
  • Clustered
  • Regularly
  • Interspaced
  • Short
  • Palindromic
  • Repeats
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23
Q

CRISPR DNA sequences act as a ______

A

Primitive immune system

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24
Q

CRISPR DNA sequences provide protection from ______ from viruses

A

Prokaryotes

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25
Q

What are CRISPR repeats?

A

Stretches of palindromic DNA

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26
Q

How long are CRISPR repeats?

A

25 - 50 bp long

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27
Q

What are CRISPR spacers?

A

Sequences from viruses that invaded the prokaryote

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28
Q

How long are CRISPR spacers?

A

20 - 80 bp long

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29
Q

Describe the process in which bacteria acquire DNA sequences (2)

A
  • A bacteriophage infects the cell
  • A piece of viral DNA is added as a spacer
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30
Q

Describe the process in which CRISPR RNAs (crRNAs) mediate defense against viral infections (2)

A
  • The spacer is transcribed into crRNA
  • crRNA guides Cas to the bacteriophage
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31
Q

What specific role do CRISPR-associated enzymes (Cas) play in the defense against pathogens / future diseases?

A

Disables the virus by cutting its DNA

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32
Q

Describe how CRISPR technology can be used in so-called ‘genome editing’

A

RNA identifies a specific piece of DNA that is inserted into a new strand

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33
Q

By whom, and in what year, was the polymerase chain reaction (PCR) developed?

A

Kary Mullis in 1983

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34
Q

Name the 3 steps that make up one cycle of a typical polymerase chain reaction

A
  • Denaturation
  • Annealing
  • Extension
35
Q

Describe the denaturation step of the polymerase chain reaction (3)

A
  • Double-stranded DNA is heated to 95°C
  • Hydrogen bonds break
  • Single-stranded templates are produced
36
Q

Describe the annealing step of the polymerase chain reaction (2)

A
  • Temperature is lowered to 45°C
  • Primers anneal to the single-stranded template
37
Q

Describe the extension step of the polymerase chain reaction (2)

A
  • Temperature is heated to 72°C
  • DNA polymerase synthesizes new strands
38
Q

Regarding PCR, what are primers?

A

Short fragments of single-stranded DNA

39
Q

Primers are ______ to known sequences

A

Complementary

40
Q

How long are primers?

A

20 nucleotides long

41
Q

In PCR, primers replicate ______ of a cell’s DNA

42
Q

In normal DNA replication, primers replicate ______ of a cell’s DNA

43
Q

What are the functions of deoxyribonucleotide triphosphates (dNTPs) in PCR? (2)

A
  • Expand the growing DNA strand
  • Bind with the cDNA strand
44
Q

At the end of one cycle of PCR, how many new double-stranded DNA molecules are produced for each original molecule of target DNA?

45
Q

At the end of one cycle of PCR, the amount of target DNA ______

46
Q

Does PCR result in a linear or an exponential increase in the amount of target DNA?

A

Exponential

47
Q

Approximately how long does PCR take to perform?

A

A few minutes

48
Q

Name the 2 key innovations that facilitated the routine use of PCR in the laboratory

A
  • Thermostable DNA polymerase
  • PCR machines
49
Q

Why was DNA polymerase from E. coli abandoned in favor of a more thermostable DNA polymerase? (2)

A
  • It denatures at 90°C
  • Fresh enzyme had to be added in each cycle which slowed the process
50
Q

What is the name of the thermostable DNA polymerase used in most modern PCR applications?

A

Taq polymerase

51
Q

How do PCR machines (thermal cyclers) work?

A

Cause temperature changes necessary for the different steps of PCR

52
Q

What does the term electrophoresis literally mean?

A

To carry with electricity

53
Q

How is electrophoresis used in molecular biology?

A

Separates molecules based on their size and electrical charge

54
Q

What type of electrophoresis is used to separate DNA molecules?

A

Gel electrophoresis

55
Q

Describe agarose (2)

A
  • Purified extract of agar isolated from seaweed
  • Melted in a buffer solution
56
Q

What is the function of agarose in electrophoresis?

A

Small pores allow DNA to migrate through

57
Q

To which end of the gel are the DNA samples added?

A

Negative end

58
Q

Describe the principle by which DNA fragments migrate through an agarose gel during gel electrophoresis

A

Since the phosphate group of each DNA carries a negative charge, the DNA fragments migrate toward the positive end of the gel

59
Q

What effect does fragment size have upon the migration rate of DNA molecules through an agarose gel during gel electrophoresis?

A

Small DNA fragments migrate more rapidly than large DNA fragments

60
Q

Comparing migration distance of the unknown fragments with the distance traveled by the size standards determines …

A

The approximate size of the unknown fragments

61
Q

How are DNA fragments visualized after gel electrophoresis?

A

Using a dye specific for nucleic acids

62
Q

Describe the role of ethidium bromide in the visualization of DNA fragments after gel electrophoresis (3)

A
  • Wedges itself between the bases of DNA
  • Fluoresces when exposed to UV light
  • Produces bands on the gel
63
Q

Describe electroporation as a means of introducing DNA into target cells

A

An electric current punctures microscopic holes through the cell membrane

64
Q

When inserting DNA into the cells of plants, algae, and fungi, they must first be converted into ______

A

Protoplasts

65
Q

What are protoplasts?

A

Cells with cell walls that have been enzymatically removed

66
Q

How are protoplasts used in electroporation? (2)

A
  • Cytoplasmic membranes of protoplasts fuse
  • Forms a cell with genomes of both parent cells
67
Q

Describe protoplast fusion as a means of generating recombinant cells

A

Recombination of DNA from 2 fused cells

68
Q

What role does polyethylene glycol (PEG) play in protoplast fusion?

A

Increases the rate of fusion

69
Q

What are gene guns?

A

Compressed helium gas that fires into a target cell

70
Q

What kinds of energy are used to power gene guns?

A

Tungsten / gold beads coated with DNA

71
Q

Microinjection is the insertion of DNA into target cells using a ______

A

Glass micropipette

72
Q

Describe heat shock as a means of introducing DNA into target cells

A

Bacterial cells are shocked into taking up DNA from their environment

73
Q

Define traditional agriculture

A

Controlled breeding of plants and animals to obtain desired traits

74
Q

Regarding selection, what is traditional agriculture based on?

75
Q

Why does selection in traditional agriculture affect many genes?

A

It is imprecise

76
Q

DNA-based techniques manipulate …

A

Multiple genes at a time

77
Q

Organisms with altered genes / altered expression of their own genes are called ______

78
Q

Describe golden rice as an example of a genetically modified crop

A

Uses genes from corn and bacteria to produce 23x as much β carotene

79
Q

β carotene is a ______

A

Vitamin A precursor

80
Q

Define bioremediation

A

Release of bacteria with the ability to detoxify pollutants

81
Q

How is bioremediation a genetic technology?

A

Organisms metabolize substances that would normally be considered toxins

82
Q

Bioremediation uses ______

A

Unaltered organisms

83
Q

Bioremediation transfers ______ to other species

A

Detox genes

84
Q

______ can more easily penetrate a polluted area

A

Protein products

MICRB 106 - Exam 3 (2 decks)

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