Lecture 15

Question 1

What do reverse genetics seek to find?

Answer 1

phenotypes linked to specific sequences of DNA (including genes)

Question 2

Producing mutations in a specific gene may reveal phenotypes that give a clue as to its function(s):

Answer 2

• Alter gene in vitro
• Introduce into cell
• Determine phenotypic effects

Question 3

Alter gene in vitro: Get it synthesised, use recombinant DNA techniques – site – directed mutagenesis

Answer 3

• Gene in plasmid
• Synthesis a primer that can hybridise to it but has a misbase mismatch responding to the mutation you want to introduce
• DNA polymerase to synthesis DNA
• Introduce into ecoli and allow replication
• Isolate desired clone

Question 4

Describe introduce DNA into cell

Answer 4

•	Direct uptake of DNA 
•	Incubate DNA with competent cells 
•	-  Bacterial/yeast transformation 
•	-  Animal cell transfection 
•	Electroporation Microinjection Virus-mediated Ballistic 
•	Gene gun 
•	Cells with walls e.g. plants Agrobacterium tumefaciens-mediated 
•	Plants and some fungi 
•	Fate of the transgene 
•	Transient expression 
•	Replicates on a plasmid 
•	Chromosomal integration 
-	Random 
-	Targeted to a particular locus 
•	Cell type 
•	Somatic or germ cell 
•	Haploid or diploid Detection of transgenic cells 
•	Selectable marker gene 
•	Dominant or recessive

Question 5

Describe gene disruption

Answer 5

• Introduce circular homologous DNA
• Select for cells expressing marker
• Single crossover

Question 6

Describe gene deletion

Answer 6

• Introduce linear homologous DNA
• Select for cells expressing marker
• Double crossover

Question 7

Describe CRISPR

Answer 7

• Clustered Regularly Interspaced Short Palindromic Repeats
• Mediated immunity in bacteria:
o Short viral DNA sequence is integrated into CRISPR locus
o CRISPR locus is transcribed and is processed and combined with a CAS protein which is an enzyme that can cut DNA – seek out DNA from new virus
o RNA base pairs with the DNA and nuclease attacks the DNA and cleaves it
• Express cas9 protein and guide RNA in the cell at the same time a double strand break will occur and the cell will repair it which could lead to mutations
o Adapt enzyme by attachig activation domain (gene on) or repressor domain (gene off)
• Not 100%, off target effects are possible
• Combined with gene insertion it enhances homologous recombination
• Ethical concerns with regard to humans

Question 8

Targeted regulated expression of native genes:

Answer 8

• Replace native promotor for your favourite gene with an active one or regulatable promoter

Question 9

Promoter activity :

Answer 9

• Replace promoter with a fluroscent marker gene and then monitor how much the promoter is turned on

Question 10

Describe analysis

Answer 10

Tag a protein – not replace it but add a DNA sequence
Introduce transgenes into animals – produces dominant phenotype
High throughput – analysis many genes at one time