Lecture 20 - Genetic Strategies

Question 1

what is forward genetics?

Answer 1

an approach that seeks to find the genes encoded by DNA that are responsible for a phenotype of interest

Question 2

what are the 3 main steps of forward genetics?

Answer 2

1. isolate mutants with a phenotype of interest
2. clone/identify the genes
3. analyse the genes to predict encoded proteins

Question 3

what are some common genetic model organisms?

Answer 3

C.elegans, D.melanogaster, E.coli, M.musculus (mice)

Question 4

how is mutagenesis done?

Answer 4

physical mutagens (UV/ionising radiation), chemical mutagens (base analogues, alkylating agents) and biological mutagens (transposable elements)

Question 5

what is an example of a conditional lethal point mutant?

Answer 5

temperature sensitive mutants that function at the permissive temperature and don’t at the non-permissive (restrictive temperature)

Question 6

what is mutant rescue/complementation?

Answer 6

transforming cells with gene/cDNA library to restore a wild-type phenotype

Question 6

how is cDNA expressed in cells?

Answer 6

it is introduced into the DNA then replicated and expressed in the cell

Question 7

how is cDNA made?

Answer 7

1. RNA is extracted
2. reverse transcriptase makes a DNA copy
3. treat with RNAse
4. make second strand with DNA polymerase

Question 8

what does BLAST stand for?

Answer 8

Basic Local Alignment Search Tool

Question 9

what is reverse genetics?

Answer 9

beginning with a particular gene and mutating it to create mutant cells/organisms to analyse gene function

Question 10

what is the basic process of reverse genetics?

Answer 10

1. altering the gene in vitro
2. introducing it into the cell
3. determining the phenotypic effects

Question 11

what are 4 ways to alter a gene?

Answer 11

1. gene knockout
2. replace with one that is expressed in the wrong tissue/at the wrong time (alters promoter)
3. engineered to be deleted in certain cell types/tissues (inducible knockout)
4. slight changes in protein structure to dissect which are important for function

Question 12

what are 5 methods to induce cell DNA uptake?

Answer 12

1. electroporation
2. microinjection
3. virus-mediated
4. ballistic (gene gun)
5. agrobacterium tumefaciens mediated

Question 13

what does CRISPR stand for?

Answer 13

Clustered Regularly Interspaced Short Palindromic Repeats

Question 14

what is the 3 step process of CRISPR?

Answer 14

1. short viral DNA sequence is intergrated into the CRISPR locus
2. RNA is transcribed from CRISPR locus, processed and bound to Cas protein
3. small crRNA in complex with Cas seeks out and destroys viral sequences

Question 15

what can CRISPR/Cas9 be used for?

Answer 15

1. disrupt or knockout a gene/sequence of choice
2. add an altered version of the gene by harnessing homologous recombination for DNA damage repair

Question 16

how may off-target effects be limited?

Answer 16

• in silico prediction of possible binding sites
• in vitro detection
• in vivo detection

Question 17

how else can proteins function be studied?

Answer 17

tagging with fluorescent protein may be able to detect cancerous cells