Genetic Screens

Question 1

Forward vs Reverse

Answer 1

Forward = genes important for process
Reverse = process gene important for

Question 2

Selection vs Screen

Answer 2

Selection = only variant survives
Screen = look for variant with phenotype

Question 3

Non-complementation

Answer 3

Same Gene

- no synthesis of leucine

Question 4

Complementation

Answer 4

Different Genes

- synthesis of leucine

Question 5

Zygotic

Answer 5

• zygote homozygous for mutation

- F3 = dead embryo

Question 6

Maternal

Answer 6

• mother homozygous for mutation –> normal egg

- F4 = larvae affected

Question 7

Transformation

Answer 7

Plasmids (have selectable marker)
Random Insertion
Homologous Recombination

Question 8

Injecting DNA

Answer 8

Random Insertions

Question 9

Transposon Mediated Transformation

Answer 9

• transposase cut transposon out of genome
• transposon contains inverted repeats
• 2 plasmids for control (1 = transposase, 2 = transposon)

Question 10

Site Specific Recombination

Answer 10

• use recombinase find recombination site

- duplicating recombination sites

Question 11

Functional Complementation

Answer 11

• DNA fragment & cdc42 lf = no complementation

- cdc42+ & cdc42 lf = complementation (growth)

Question 12

Mutational Analysis

Answer 12

• mutation in gene
• which mutations complement cdc42lf allele
• important for function

Question 13

Misexpression

Answer 13

• heat shock promoter express ANTP all cells

Question 14

Knock Out Genes

Answer 14

• homologous recombination

- interrupts gene

Question 15

RNAi

Answer 15

• dicer cuts into 20 nucleotide fragments
• binds to AGO (RNA induced silencing complex)
• binds and degrades mRNA

Question 16

lin-14

Answer 16

lin-14 lf = advanced development L2

lin-14 gf = suppress L2 + replace with L1

Question 17

lin-4

Answer 17

• regulates expression of lin-14

- SUPPRESS translation of lin-14 in L2

Question 18

miRNA

Answer 18

• in genome –> pri-miRNA —> pre-miRNA
• dicer cuts up
• binds to AGO (RNA induced silencing complex)
• suppress translation
• miRNA guides RISC to specific mRNA

Question 19

RNAi Screens

Answer 19

1) insert cDNA of gene into E.coli plasmid
2) induce expression = RNA copy
3) C.elegans eat E.coli –> dsRNA absorbed
4) dsRNA diced, bind to AGO, degrades = no translation

Question 20

Restriction Modification Immunity

Answer 20

1) Restriction enzyme cuts phage DNA

2) Methylase methylates genomic DNA protect from restriction enzyme

Question 21

CRISPR (Acquire)

Answer 21

1) CAS cuts phage DNA next to PAM sequence (NGG)

2) Inject phage DNA into genomic DNA

Question 22

CRISPR (Immunity)

Answer 22

1) CRISPR array –> pre-crRNA
2) Cas9 (guide RNA + tracer RNA) made
3) Cas9 finds PAM in phage DNA
4) guide RNA unwinds dsDNA –> ds break = inactive

NO PAM = cannot cut genomic DNA

Question 23

CRISPR (Reengineered)

Answer 23

1) Cas9 (single mRNA with guide function + seq to bind)
2) Creates ds breaks

• attach function to Cas9
• mutations to knockout ability make ds break
• find and bind to unique sequences in genome