Genetic Screens Flashcards
Forward vs Reverse
Forward = genes important for process Reverse = process gene important for
Selection vs Screen
Selection = only variant survives Screen = look for variant with phenotype
Non-complementation
Same Gene
- no synthesis of leucine
Complementation
Different Genes
- synthesis of leucine
Zygotic
- zygote homozygous for mutation
- F3 = dead embryo
Maternal
- mother homozygous for mutation –> normal egg
- F4 = larvae affected
Transformation
Plasmids (have selectable marker)
Random Insertion
Homologous Recombination
Injecting DNA
Random Insertions
Transposon Mediated Transformation
- transposase cut transposon out of genome
- transposon contains inverted repeats
- 2 plasmids for control (1 = transposase, 2 = transposon)
Site Specific Recombination
- use recombinase find recombination site
- duplicating recombination sites
Functional Complementation
- DNA fragment & cdc42 lf = no complementation
- cdc42+ & cdc42 lf = complementation (growth)
Mutational Analysis
- mutation in gene
- which mutations complement cdc42lf allele
- important for function
Misexpression
- heat shock promoter express ANTP all cells
Knock Out Genes
- homologous recombination
- interrupts gene
RNAi
- dicer cuts into 20 nucleotide fragments
- binds to AGO (RNA induced silencing complex)
- binds and degrades mRNA
lin-14
lin-14 lf = advanced development L2
lin-14 gf = suppress L2 + replace with L1
lin-4
- regulates expression of lin-14
- SUPPRESS translation of lin-14 in L2
miRNA
- in genome –> pri-miRNA —> pre-miRNA
- dicer cuts up
- binds to AGO (RNA induced silencing complex)
- suppress translation
- miRNA guides RISC to specific mRNA
RNAi Screens
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
Restriction Modification Immunity
1) Restriction enzyme cuts phage DNA
2) Methylase methylates genomic DNA protect from restriction enzyme
CRISPR (Acquire)
1) CAS cuts phage DNA next to PAM sequence (NGG)
2) Inject phage DNA into genomic DNA
CRISPR (Immunity)
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
CRISPR (Reengineered)
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
