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Microbiology1 > Ch 9: Biotechnology and Recombinant DNA > Flashcards

Ch 9: Biotechnology and Recombinant DNA Flashcards

1
Q

What is the function of restriction enzymes?

A
  • Cut DNA at defined positions close to or within their recognition sequence
  • Typically recognize 4, 6, or 8 base sequences
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2
Q

True or false. Blunt ends of DNA can only ligate with specific blunt ends.

A

False. Any blunt ends can ligate together

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3
Q

True or false. Stick-end DNA overhangs must be complementary in order to ligate.

A

True

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4
Q

Why do bacteria produce restriction enzymes?

A

They restrict the ability of foreign DNA (such as bacteriophage DNA) to infect/invade the host cell by cleaving it

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5
Q

How is host DNA modified to protect it from its own restriction enzymes?

A

Methylation of their sequences at C or A nucleotides

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6
Q

What E. coli plasmid vector is commonly used for cloning?

A

pUC19

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7
Q

What protein does the gene lacZ encode for?

A

β-galactosidease

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8
Q

What protein does the gene ampR encode for?

A

Ampicillin resistance

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9
Q

What stain is used to visualize DNA on an agarose gel?

A

Ethidium Bromide

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10
Q

What are some methods of transformating DNA into cells in the lab? (5)

A
  1. Electroporation
  2. Chemical transformation
  3. Protoplast fusion
  4. Gene gun
  5. Microinjection
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11
Q

What is the difference between transformation and transfection?

A

Transformation: uptake of DNA into a prokaryotic cell; term means something different in mammalian cells

Transfection: uptake of DNA into mammalian cells

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12
Q

True or false. Most cell types don’t naturally transform.

A

True

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13
Q

How does electroporation work?

A
  • Controlled short, but powerful electrical pulse induces temporary hydrophilic pores in in the cell membrane
  • DNA can enter the cell
  • Applicable to all cells
  • Organisms with cell walls may require conversion to protoplast
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14
Q

Explain how chemical transformation works in E. coli

A
  • Cells incubated in ice-cold calcium chloride
  • DNA is added
  • Mild heat shock
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15
Q

Explain how chemical transformation works for yeast

A
  • Incubated in ice-cold lithium chloride
  • DNA added
  • Mild heat shock
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16
Q

Rates of protoplast fusion increase in the presence of _____

A

Polyethyleneglycol (PEG)

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17
Q

How does a gene gun (or biolistic transformation) work?

A
  • Microscopic gold or tungsten particles (1 μm) coated with DNA propelled into cells with burst of helium
  • If a particle lands on the nucleus the DNA may be incorporated into the chromosome
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18
Q

In what organisms is biolistic transformation typically used? (3)

A
  1. Plant cells
  2. C. elegans
  3. Yeast mitochondria
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19
Q

How does microinjection for transformation work?

A
  • Uses glass micropipette to inject DNA into the cell
20
Q

True or false. Microinjection is most commonly used for bacterial and fungal transformations.

A

False. Bacteria and fungi are too small. This method is impractical.

21
Q

What is a DNA library?

A

A collection of DNA fragments of one organism, each carried by a plasmid or virus and cloned in an appropriate host

22
Q

What are the two types of DNA libraries?

A
  1. Genomic library: DNA fragments representing an entire organism’s genome
  2. cDNA library: only complementary DNA molecules synthesized from mRNA molecules in a cell
23
Q

What nucleotide sequence does the restriction enzyme Sau3A recognize?

A

GATC

24
Q

In genomic library construction, how many genome equivalents are necessary to ensure that all genomic sequences will be represented with 95% probability?

A

5 genome equivalents

25
Q

In genomic library construction, how many genome equivalents are necessary to ensure that all genomic sequences will be represented with 99% probability?

A

10 genome equivalents

26
Q

cDNA is made from mRNA by what enzyme?

A

Reverse transcriptase

27
Q

What are oligonucleotides (ssDNA)?

A

Synthetic DNA that can be made chemically (not enzymatically) on a solid matrix using a DNA synthesis machine

28
Q

What are some limitations of ssDNA?

A
  • Limited to less than 200 bases in length
  • Gene sequence will be ambiguous when only the protein sequence is available
29
Q

What cell types are commonly used to make gene products?

A
  1. E. coli
  2. Saccharomyces cerevisiae
  3. Plant cells and whole plants
  4. Mammalian cells
30
Q

What are some disadvantages of using E. coli to make gene products?

A
  1. Need to eliminate endotoxins from the products, since E. coli is gram-negative
  2. Does not normally secrete products; need to lyse the cells to get products, which is expensive on an industrial scale
31
Q

What bacteria is alternatively used instead of E. coli to harvest gene products via secretion rather than lysing?

A

Bacillus subtilis

32
Q

What are some advantages of using Saccharomyces cerevisiae to make gene products? (4)

A
  1. Easily grown with well-understood genomics
  2. Can carry plasmids
  3. Greater chance of expressing and correctly modifying eukaryotic genes and proteins
  4. Likely to continuously secrete products
33
Q

What are some advantages of using plant cells to make gene products? (3)

A
  1. May express eukaryotic genes easily
  2. Low risk of product contamination by mammalian pathogens
  3. Large scale low-cost production
34
Q

What are some advantages of using mammalian cells to make gene products? Disadvantage?

A

Adv:

  1. May express eukaryotic genes easily
  2. Well suited to make proteins for medical use; secreted and low risk of toxins

Disadv:

  1. Harder to grow
35
Q

What are some therapeutic applications of rDNA?

A
  1. Human enzymes and other proteins (insulin, etc.)
  2. Subunit vaccines
  3. DNA vaccines
  4. Gene therapy
36
Q

What is gene therapy?

A

The introduction, removal, or change in genetic material in the cells of a patient to treat an inherited or developed disease.

37
Q

Insulin’s subunits are composed of _____ amino acids.

A

21 - 30

(per subunit, insulin has 2)

38
Q

What vector is showing promise for use in gene therapy?

A

Adenovirus vectors

39
Q

_______ is a eukaryotic biological process in which RNA molecules inhibit gene expression

A

RNA intererence (RNAi) or gene silencing

40
Q

What is the difference between gene editing and gene therapy?

A

Editing: remove, disrupt, or correct faulty elements of DNA within the gene

Therapy: replaces the entire gene

41
Q

What 3 components are required for gene editing?

A
  1. Cas9
  2. Guide RNA
  3. New designed DNA
42
Q

CRISPR stands for:

A

clustered regularly interspaced short palindromic sequences

43
Q

_____% of the human genome encodes for product, while the remainder is “junk DNA”

A

2%

44
Q

What are some of the components of “junk DNA” in the human genome? (7)

A
  1. miRNA genes
  2. Short tendem repeats
  3. Introns
  4. Telomeres
  5. Transposons
  6. Viral remnants
  7. Pseudogenes
45
Q

_____ is the science of understanding gene function through computer assisted analysis.

A

Bioinformatics

46
Q

The bacterium _______ infects specific plants at wound sites with plasmid DNA (Ti plasmid) that contains genes for opine synthesis and tumor production (crown gall)

A

Agrobacterium tumefaciens

Microbiology1 (20 decks)

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