Reverse Genetics Flashcards

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1
Q

Give the definition of reverse genetics.

A

Reverse genetics = seeks to find the phenotypes linked to specific sequences of DNA.

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2
Q

Give the process of reverse genetics.

A
  1. Alter gene in vitro
    -get altered sequence synthesised or use recombinant DNA techniques
  2. Introduce into cell
    -direct uptake
    Incubate DNA with competent cells
    Bacterial/yeast transformation
    Animal cell transfection
-aid/induce uptake
Electroporation
Microinjection
Virus-mediated
Ballistic (gene gun or cells with walls)
Agrobacterium tumefaciens-mediated (plants and some fungi)

-fate of the transgene
Transient expression
Replicates on a plasmid
Chromosomal integration (random or targeted to a particular locus)

-cell type
Somatic or germ
Haploid or diploid

-detection of transgenic cells
Selectable marker gene
Yeast & bacteria often use auxotrophic mutants

  1. Determine phenotypic effects
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3
Q

Describe how CRISPR is used for gene editing.

A
  • CRISPR-mediated viral defence in bacteria
  • bacteria cleave off small sequence from DNA of an infecting virus that they have destroyed and integrate into their own bacterial genome
  • this DNA is transcribed from the CRISPR locus, processed and bound to Cas protein
  • easily altered guide RNAs can guide the Cas9 endonuclease to a specific gene
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4
Q

Describe promoter activity reporters & limitations of using them.

A
  • clone reporter after promoter sequence, replacing native ORF Level detected should reflect promoter activity
  • most commonly used is Green flurorescent protein (GFP)
  • runs into problems with haploid organisms, complex promoters, or organisms with no formal genetics
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5
Q

Give the purpose of protein localisation & movement.

A

Tag protein with GFP so when it is incorporated it will fluoresce and so we can see it.

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6
Q

Describe the process of RNA interference.

A
  • introduce double strands of DNA into cells, they become incorporated into argonaute or Piwi proteins to target RNAs produced by cell
  • specific knockdown of gene expression
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7
Q

Why is RNAi used as experimental tool for specific gene knockdown?

A
  • applicable to many eukaryotes

- individual gene knockdown

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8
Q

Why are high throughput screens required?

A

Organisms are complex.

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