reverse genetics

Question 1

reverse genetics approaches to seek

Answer 1

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

Question 2

how may the gene responsible for a certain phenotype be revealed

Answer 2

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

Question 3

reverse genetics method simple (3)

Answer 3

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

Question 4

alter gene in vitro

Answer 4

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

Question 5

site directed mutagenesis

Answer 5

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

Question 6

introduce DNA into ells

Answer 6

• direct uptake of DNA
• electroportation
• agrobacterium tumefaciens-mediated

Question 7

direct uptake of DNA

Answer 7

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

Question 8

transfection

Answer 8

animal cells

Question 9

transformation

Answer 9

yeast and bacteria

Question 10

electroporation

Answer 10

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

Question 11

ballistic

Answer 11

gene gun cells with walls e.g. plants

Question 12

gene gun is for cells with

Answer 12

walls e.g. plants

Question 13

agrobacterium tumefaciens mediated

Answer 13

plants and some fungi

Question 14

Fate of the trangene

Answer 14

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

Question 15

DNA can be introduced to four different types of cells

Answer 15

somatic germ haploid diploid

Question 16

how are transgenic cells detected

Answer 16

using selectable marker genes or dominant or recessive nature

Question 17

what can be utilised for gene targeting

Answer 17

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

Question 18

targeted gene disruption by homologous recombination

Answer 18

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

Question 19

targeted gene deletion by homologues recombination

Answer 19

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

Question 20

targeted gene deletion of diploid yeast requires..

Answer 20

two marker genes or a marker recycling system

Question 21

marker cycling scheme

Answer 21

.

Question 22

deleted genes can be replaced with a..

Answer 22

mutant gene and selectable marker by homologous recombination

Question 23

targeted gene deletion of a diploid animals..

Answer 23

more complex

Question 24

CRISPR

Answer 24

Clustered regularly interspaced short palindromic repeats

Question 25

CRISPR-mediated immunity bacteria

Answer 25

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

Question 26

CRISPR/ Cas9 for gene activation/repression

Answer 26

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

Question 27

CRISPR is not

Answer 27

100%; off target effects are possible

Question 28

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

Answer 28

CRISPR/Cas9 for gene editing

Question 29

targeted regulated expression of native genes

Answer 29

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

Question 30

targeted regulated expression of native genes

Answer 30

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

Question 31

promotor activity effects

Answer 31

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

Question 32

what can be used to determine promotor activity

Answer 32

reporters

Question 33

example of reporters used to determine promotor activity

Answer 33

B-Galactosidase, Lucifer's, GFP

Question 34

promotor activity- reporters : process

Answer 34

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

Question 35

ORF

Answer 35

open reading frame

Question 36

ORF

Answer 36

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

Question 37

protein localisation and movement

Answer 37

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

Question 38

GFP

Answer 38

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.)

Question 39

how can we avoid genomic editing using RNAi

Answer 39

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

Question 40

'knockdown' of gene expression

Answer 40

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

Question 41

'knockdown' of gene expression

Answer 41

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

Question 42

we need high throughput technologies because..

Answer 42

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

Question 43

examples of high throughput technologies

Answer 43

pic

Question 44

high throughput reverse genetic study using C.elegans RNAi

Answer 44

- 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

Question 45

dsRNA

Answer 45

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

Question 46

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

Answer 46

screened separately

Question 47

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

Answer 47

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

Question 48

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

Answer 48

screened together by competition

Question 49

in conclusion reverse genetics

Answer 49

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