L10 Mutagenesis

Question 1

Forward genetics

Answer 1

Phenotype => genotype

• random mutagenesis
• screen
• identify gene

Question 2

Reverse genetics

Answer 2

genotype => phenotype

• candidate gene
• targeted mutagenesis
• mutant
• observation
• define phenotype

Question 3

Chemical mutagenesis - EMS

Answer 3

Ethyl methanesulfonate (EMS)

• adds ethyl group onto guanine
• ethyguanine pairs with thymine => mutation
• induce point mutaition

GC => AT transistion

Question 4

Chemical mutagenesis - ENU

Answer 4

N-ethyl-N-nitrosourea

• adds ethyl group to thymine
• AT => GC transition
• point mutation

Question 5

Missense

Answer 5

In genetics, a missense mutation is a point mutation in which a single nucleotide change results in a codon that codes for a different amino acid. It is a type of nonsynonymous substitution.

Question 6

Nonsense

Answer 6

a mutation in which a sense codon that corresponds to one of the twenty amino acids specified by the genetic code is changed to a chain-terminating codon.

Question 7

Physical mutagenesis

Answer 7

• ionising radiation
• induce substitutions, ss breaks, DSB, indels, missense, nonsense
• random

Question 8

Insertional mutagenesis - transposons

Answer 8

• transposons

DNA transposons

• autonomous/non-autonomous
• random insertion
• indel from imperfect Tn excision
• gene disruption by Tn insertion
• insertion can lead to complete or partial loss of gene expression

Question 9

Insertional mutagenesis - T-DNA

Answer 9

transfer-DNA
- agrobacterium derived tumour-induced plasmid

T-DNA insertions

• plant transformation
• random insertion

Tn and T-DNA can be engineered e.g. selectable markers, reporter genes, ehancer

Question 10

Targeted mutagenesis - RNAi

Answer 10

• knock down of gene expression
• requires microculture assay
• not heritable, not genetic
• requires sequence information to design sgRNA

Question 11

Targeted mutagenesis -CRISPR/Cas9

Answer 11

• induces insertions, deletions, point mutations

- requires sequence information to design gRNA

Question 12

Hypomorphic allele

Answer 12

Hypomorphic describes a mutation that causes a partial loss of gene function. A hypomorph is a reduction in gene function through reduced (protein, RNA) expression or reduced functional performance, but not a complete loss

Question 13

Genetic screen

Answer 13

A genetic screen or mutagenesis screen is an experimental technique used to identify and select for individuals who possess a phenotype of interest in a mutagenized population. Hence a genetic screen is a type of phenotypic screen.

Question 14

Genetic selection

Answer 14

only the desired mutation survives

Question 15

Circadian clock

Answer 15

A circadian clock, or circadian oscillator, is a biochemical oscillator that cycles with a stable phase and is synchronized with solar time.

Question 16

Phenotyping strategies - reported based screens

Answer 16

• generate transgenic organisms to report specific process or pathway of interest
• histochemical (lacZ), luminescent (luciferase), fluorescent (GFP)

Question 17

Phenotyping strategies - modifier screens

Answer 17

• mutations that alter then mutant phenotype are likely to affect the same molecular pathway

Question 18

Mapping insertion mutations

Answer 18

• insertion sequence known, mapping by inverse PCR

Question 19

Mapping point mutations

Answer 19

• principle for map-based cloning
• mapping strain contains sufficient genetic variation from the strain containing the mutation to differentiate alleles across the genome