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Flashcards in reverse genetics Deck (49):
1

reverse genetics approaches to seek

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

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how may the gene responsible for a certain phenotype be revealed

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

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reverse genetics method simple (3)

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

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alter gene in vitro

-first get it synthesised

-use recombinant DNA techniques e.g. site directed mutagenesis

e.g. using restriction endonucleases

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site directed mutagenesis

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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introduce DNA into ells

-direct uptake of DNA

-electroportation

-agrobacterium tumefaciens-mediated

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direct uptake of DNA

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

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transfection

animal cells

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transformation

yeast and bacteria

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electroporation

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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ballistic

gene gun cells with walls e.g. plants

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gene gun is for cells with

walls e.g. plants

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agrobacterium tumefaciens mediated

plants and some fungi

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Fate of the trangene

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

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DNA can be introduced to four different types of cells

somatic germ haploid diploid

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how are transgenic cells detected

using selectable marker genes or dominant or recessive nature

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what can be utilised for gene targeting

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

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targeted gene disruption by homologous recombination

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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targeted gene deletion by homologues recombination

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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targeted gene deletion of diploid yeast requires..

two marker genes or a marker recycling system

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marker cycling scheme

.

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deleted genes can be replaced with a..

mutant gene and selectable marker by homologous recombination

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targeted gene deletion of a diploid animals..

more complex

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CRISPR

Clustered regularly interspaced short palindromic repeats

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CRISPR-mediated immunity bacteria

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

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CRISPR/ Cas9 for gene activation/repression

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

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CRISPR is not

100%; off target effects are possible

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

CRISPR/Cas9 for gene editing

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targeted regulated expression of native genes

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

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targeted regulated expression of native genes

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

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promotor activity effects

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

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what can be used to determine promotor activity

reporters

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example of reporters used to determine promotor activity

B-Galactosidase, Lucifer's, GFP

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promotor activity- reporters : process

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

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ORF

open reading frame

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ORF

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

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protein localisation and movement

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

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GFP

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.)

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how can we avoid genomic editing using RNAi

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

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'knockdown' of gene expression

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

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'knockdown' of gene expression

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

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we need high throughput technologies because..

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

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examples of high throughput technologies

pic

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high throughput reverse genetic study using C.elegans RNAi

-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

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dsRNA

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

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

screened separately

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

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

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

screened together by competition

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in conclusion reverse genetics

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