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BIO1334 - Genetics > Genetic Strategies > Flashcards

Genetic Strategies Flashcards

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1
Q

What is the forward genetic approach?

A

Seek to find the genes encoded by DNA that are responsible for a phenotype of interest

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2
Q

Summaries the forward genetic approach:

A
  • Starts with phenotype
  • A series of mutants with defects in a particular phenotype may be linked in the same molecular pathways/functions
  • Enables the wild-type genes for this pathway to be identified and studied
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3
Q

Describe the forward genetic approach:

A

1) Isolate mutants with a phenotype of interest.
2) Define genes responsible for these phenotypes.
3) Clone/identify the genes.
4) Analyse the genes to predict encoded proteins

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4
Q

Describe the isolate mutants with a phenotype of interest stage of forwards genetics of forward genetics:

A
  • Choose good organism for genetics
  • Mutagenise (physical, chemical and biological mutagens)
  • Screen for mutants with the desired phenotype (For essential cell functions require conditional lethal point mutants)
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5
Q

Examples of genetic model organisms:

A
  • Saccharomyces cerevisiae
  • Caenorhabditis elegans
  • Drosophila melanogaster
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6
Q

What are physical mutagens?

A
  • UV

- Ionising radiation

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7
Q

What are chemical mutagens?

A
  • Base analogous

- Alkylating agents

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8
Q

What are biological mutagens?

A

Transposable elements

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9
Q

What are screen for the desired phenotype?

A

Temperature sensitive mutants (mutant protein is functional at the permissive temperature/non-functional at the non-permissive (restrictive) temperature)

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10
Q

Give an example forward genetics:

A

Cell cycle - fission yeast Schizosachhoromyces pombe

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11
Q

Describe define genes responsible for these phenotypes stage of forwards genetics:

A

Genetic complementation tests can determine if a phenotype arises from mutations in the same or separate genes

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12
Q

Describe clone/identify the genes stage of forwards genetics:

A

Clone genes by mutant rescue (mutant complementation)
Transform cells with gene/cDNA library
Isolate transform ants which rescue, or restore a wild type phenotype

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13
Q

What is a gene library?

A

Collection of recombinant clones

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14
Q

Describe analyse the genes to predict encoded proteins stage of forward genetics:

A
  • Compare predicted protein sequence with database sequences
  • BLAST = Basic Local Alignment Search Tool
  • Gives clue to function, which can be testes
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15
Q

What is the eukaryotic cell cycle controlled by?

A

Protein phosphorylation

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16
Q

What is responsible for cell cycle control?

A

CDKs

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17
Q

How were CDKs discovered?

A

By genetic analysis

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18
Q

Which of the following sets of procedures is involved in forward genetics?

A) Alter gene in vitro; introduce into cell; determine phenotypic effects.
B) Clone gene into plasmid; hybridise mutant oligonucleotide; grow up in Escherichia coli; isolate desired mutagenised clone.
C) Isolate mutants; define genes responsible; clone the gene.
D) Transcription; RNA processing; translation.

A

C

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19
Q

What is reverse genetics approach?

A

Seeks to find the phenotypes linked to specific sequences of DNA

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20
Q

How does reverse genetics approach try and find out the phenotypes linked to specific DNA sequences?

A
  • Complete knockout the function of a gene or alter its sequence
  • Then observe the effect phenotypic affects
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21
Q

How is reverse genetics done traditional ?

A

Alteration to genes in complete vitro

22
Q

How is reverse genetics done now?

A

Deleting, disrupting or producing mutations in a specific gene may reveal phenotypes that can reveal their functions

23
Q

Summaries the mechanism to reverse genetics approach:

A

1) Alter gene in vitro
2) Introduce into cell
3) Determine phenotypic effects

24
Q

Describe alter gene in vitro stage of reverse genetics?

A
  • DNA sequences synthesised

- Use recombinant DNA techniques

25
Q

Give an example of recombinant DNA techniques are used in reverse genetics:

A

Oligonucleotide-mediated site-directed mutagenesis

26
Q

Describe Oligonucleotide-mediated site-directed mutagenesis:

A
  • Gene in plasmid
  • Denature and hybridise mutant oligo
  • Grow up in E.coli
  • Isolate desire clone
27
Q

Describe introduce DNA into cell stage of reverse genetics:

A 
Take  up in cell:
-Direct uptake of DNA
or
-Aid/Induce cell DNA uptake
When take up:
-transgene has transient expression and replicates on a plasmid
-Chromosomal interaction (random or targeted to a particular locus)
-Somatic or germ cell
-Haploid or diploid
28
Q

How is direct uptake of cell done in reverse genetics?

A

Incubate DNA with competent cells

Bacterial/yeast transformation or animal cell transfection

29
Q

How is aided/induce cell DNA uptake in reverse genetics?

A
  • Electroporation
  • Microinjection
  • Virus-mediated
  • Ballistic (Gene gun/Cells with walls e.g. plants)
  • Agrobacterium tumefaciens-mediated (Plants and some fungi)
30
Q

Hoe are transgenic cells detected in reverse genetics?

A
  • Selectable marker gene

- Yeast and bacteria often use auxotrophic mutants

31
Q

Describe phenotypic effects:

A

Compare between controlled and experimented organism

32
Q

How can homologous recombination be utilised for gene targeting?

A
  • Breakage and rejoining of DNA

- Gene disruption/deletion

33
Q

What may gene targeting need?

A

Marker genes or a marker recycling scheme

34
Q

What does CRISPR stand for?

A

Clustered Regularly Interspaced Short Palindromic Repeats

35
Q

Describe the steps of CRISPR-mediated viral defence in bacteria:

A
  • Short viral DNA sequences is integrated into CRISPR locus
  • RNA is transcribed from CRISPR locus, processed, and bound to Cas protein
  • Small crRNA in complex with Cas seeks out and destroys viral sequences
36
Q

How can CRISPR be used for gene editing?

A
  • Double strand break
  • Activation of gene
  • Repressor of gene
37
Q

What concerns are there about CRISPR and gene editing?

A

Ethical concerns in regards to humans

38
Q

What can CRISPR be used with for gene editing?

A

gene insertion

39
Q

What does CRISPR and gene insertion enhance?

A

Homologous recombination

40
Q

What does targeted regulated expression of native genes mean?

A

Replacing native promoter for your favourite gene with and active one or regulatable promoter

41
Q

What are promoter activity reporters?

A

Reporters identify when and where genes are switched on and off by the promoter

42
Q

Examples of reports:

A

β-Galactosidase, Luciferase, Green fluorescent protein (GFP) reporters

43
Q

When are reports a problem?

A

Problem for haploid organisms, problem with complex promoters, problem for organisms with no formal genetics

44
Q

Why do we tag proteins with fluorescence?

A

Allows observation of where DNA is and what it is doing and get clues of their function

45
Q

How can we avoid genomic editing?

A

By using RNAi

46
Q

What does RNAi do?

A

-Cleavage of targeting RNA
or
-Translation repression and destruction of target RNA
or
-Formation of heterochromatin on DNA from which target RNA is being transcribed

47
Q

What is RNA interference forwards or reverse genetics approach?

A

Reverse genetics approach

48
Q

What is RNAi an experimental tool for?

A

Specific gene “knockdown”

49
Q

What is high throughput?

A

Use of automated equipment to rapidly test thousands to millions of samples for biological activity at the model organism, cellular, pathway, or molecular level.

50
Q

Why do we use high throughput?

A

-Organism are complex

BIO1334 - Genetics (24 decks)

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