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Genetics > reverse genetics > Flashcards

reverse genetics Flashcards

1
Q

reverse genetics approaches to seek

A

the phenotype linked to specific sequences of DNA (including genes)

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2
Q

how may the gene responsible for a certain phenotype be revealed

A

producing mutations in a specific gene may reveal phenotypes that give a clue as to its function

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3
Q

reverse genetics method simple (3)

A

1.alter the gene in vitro 2. introduce into cell 3. determine phenotypic effect

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4
Q

alter gene in vitro

A
  • first get it synthesised
  • use recombinant DNA techniques e.g. site directed mutagenesis
    e. g. using restriction endonucleases
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5
Q

site directed mutagenesis

A

in reverse geentics oligonucleotide mediated site directed mutagenesis (SDM) is used. -plasmid is dentured and hybridised to a mutant oligo. Then this transformed plasmid is frown up in E.coli. -then the desired clone is isolated

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6
Q

introduce DNA into ells

A
  • direct uptake of DNA
  • electroportation
  • agrobacterium tumefaciens-mediated
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7
Q

direct uptake of DNA

A

incubate DNA with competent cells -bacterial/yeast transformation -animal cell transfection

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8
Q

transfection

A

animal cells

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9
Q

transformation

A

yeast and bacteria

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10
Q

electroporation

A

the action or process of introducing DNA or chromosomes into bacteria or other cells using a pulse of electricity to open the pores in the cell membranes briefly. -microinjection -virus mediated

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11
Q

ballistic

A

gene gun cells with walls e.g. plants

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12
Q

gene gun is for cells with

A

walls e.g. plants

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13
Q

agrobacterium tumefaciens mediated

A

plants and some fungi

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14
Q

Fate of the trangene

A

-transient expression -replicates on a plasmid -chromosomal integration random but can also be targeted to a particular locus

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15
Q

DNA can be introduced to four different types of cells

A

somatic germ haploid diploid

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16
Q

how are transgenic cells detected

A

using selectable marker genes or dominant or recessive nature

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17
Q

what can be utilised for gene targeting

A

homologues recombination -occurs in meiosis -breakage and rejoining of DNA -reciprocal -genetic rearrangement

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18
Q

targeted gene disruption by homologous recombination

A

Circular homologous DNA with SEL is introduced. Select for cells expressing marker. Single crossover occurs within the gene on the chromosome. Therefore it is disrupted and not expressed

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19
Q

targeted gene deletion by homologues recombination

A

linear homologous dna with SEL is introduced. Select for cells expressing marker. Gene is deleted due to a double crossover on each side of the the gene

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20
Q

targeted gene deletion of diploid yeast requires..

A

two marker genes or a marker recycling system

21
Q

marker cycling scheme

A

.

22
Q

deleted genes can be replaced with a..

A

mutant gene and selectable marker by homologous recombination

23
Q

targeted gene deletion of a diploid animals..

A

more complex

24
Q

CRISPR

A

Clustered regularly interspaced short palindromic repeats

25
Q

CRISPR-mediated immunity bacteria

A

step 1: short viral DNA sequence is integrated into CRISPR locus step 2: RNA is transcribed from CRISPR locus, processed and bound to a Cas protein step 3: small crRNA in couple with Cas seeks out and destroys viral sequence

26
Q

CRISPR/ Cas9 for gene activation/repression

A

-activation domain can attach to complex and switch gene on -repressor domain can attach and turn gene off

27
Q

CRISPR is not

A

100%; off target effects are possible

28
Q

there are ethical concerns with regard to humans when it comes to

A

CRISPR/Cas9 for gene editing

29
Q

targeted regulated expression of native genes

A

replace native promoter for YOUR FAVOURITE GENE (YFG) with a very active one, or a regulatable promotor e.g. GAL 1p -determine phenotype when over or under expressed

30
Q

targeted regulated expression of native genes

A

replace native promoter for YOUR FAVOURITE GENE (YFG) with a very active one, or a regulatable promotor e.g. GAL 1p -determine function of certain gene when phenotype is over or under expressed

31
Q

promotor activity effects

A

how much of a specific protein is being produced due to the rate of transcription

32
Q

what can be used to determine promotor activity

A

reporters

33
Q

example of reporters used to determine promotor activity

A

B-Galactosidase, Lucifer’s, GFP

34
Q

promotor activity- reporters : process

A

Clone reporters are inserted (homologous recombination) after the promotor sequence, replacing native ORF (open reading frame). –> the level of GFP protein produced will show the activity of that specific promotor. –> e.g. if lots of GFP is produced then the activity of the promotor is obviously high

35
Q

ORF

A

open reading frame

36
Q

ORF

A

open reading frame An ORF is a continuous stretch of codons that do not contain a stop codon (usually UAA, UAG or UGA).

37
Q

protein localisation and movement

A

tag with GFP -microtubules can be seen this way as well as mitochondria as as well as neurones

38
Q

GFP

A

Origin: Bioluminescent jellyfish (Aequorea victoria): Autocatalytic, fluorescent protein In vivo reporter, high signal to noise, no enzymatic activity Many spectral mutants available (RFP, YFP, BFP etc.)

39
Q

how can we avoid genomic editing using RNAi

A

instead of editing DNA, RNAi targets RNA, that is perhaps being over translated and causes its degradation -specific ‘knockdown’ of gene expression

40
Q

‘knockdown’ of gene expression

A

meaning that instead of DNA being altered the translation of RNA of a specific gene is reduced e.g. using RNAi or siRNA

41
Q

‘knockdown’ of gene expression

A

meaning that instead of DNA being altered the translation of RNA of a specific gene is reduced e.g. using RNAi or siRNA

42
Q

we need high throughput technologies because..

A

organisms are complex and processes that take a long time are expensive

43
Q

examples of high throughput technologies

A

pic

44
Q

high throughput reverse genetic study using C.elegans RNAi

A
  • each well contains E.coli expressing a different dsRNA–> target diff RNA
  • C.elegans (worm) is added to the 96 well plate
  • worms injest E.coli.
  • resulting phenotypes are recorded and analysed we can screen phenotypes separately
45
Q

dsRNA

A

dsRNA forms the genetic material of some viruses (double-stranded RNA viruses).

46
Q

during high throughput reverse genetic study using C.elegans RNAi, phenotypes can be

A

screened separately

47
Q

Genome wide screens for fitness using a large pool of barcoded yeast deletion mutants

A

a pool of barcoded yeast mutants, each deleted for a diff gene is grown in a condition of choice.

Then purified.

The relative abundance of each barcode is then recorded . -screen phenotypes together by competition

48
Q

during genome wide screens for fitness using a large pool of barcoded yeast deletion mutants phenotypes can be

A

screened together by competition

49
Q

in conclusion reverse genetics

A

seeks to find the phenotype linked to specific sequences of DNA (including genes) whereas forward genetic seeks to find the DNA sequence responsible for certain phenotypes

Genetics (24 decks)

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