Forward genetics
start with phenotype difference to identify genetic basis
reverse genetics
manipulate specific genetic change to identify its phenotypic effect
saturation mutagenesis
deliberately cause random mutations across the entire g enome of many individuals with the goal ofgetting at least one mutation in every single gene in the organism
difference between F1 and F3 screen
F1 screen identifies dominant mutations. F3 screen identifies recessive mutations
Conditional mutant analysis
helps researchers identify a mutation that is lethal in haploid systems. mutants are viable in certain conditions but non-viable in a restrictive condition
synthetic lethality
combination of two viable mutations results in an inviable double mutant
genomic library
collections of cloned DNA fragments that represent the entire genome of an organism
cDNA library
collections of cloned DNA fragments representing all mRNA produced by an organism
Complementation analysis
introduce a wt gene to the mutant and revert the mutant phenotype to the wild type
Homologous recombination
KO/knock-in at a specific locus by exchanging chromosomal DNA with donor DNA (plasmid). Cells use recombination proteins to swap DNA segments between homologous sequences.
- Homology arms: donor flanks matching the target locus
- selectable marker: sequence on the donor used to identify successful recombinants and ensure target gene integration/replacement
- because the reciprocal product is acentric, its degraded
Random insertional mutagenesis + gene-specific screening
- endogenous system: transposons insert randomly into chromosomes to build a large mutant library by random insertion
- isolate DNA from each line/pool
- PCR screen with gene primers and element-end primers
gene silencing by double-stranded RNA (RNAi)
- endogenous pathway
- long dsRNA is process by DIcer into ~21-24 nt siRNAs
- one strand leads into RISC (rna induced silencing complex) with Argonaute as the slicer
- argonaute cleaves the complementary mRNA, and it degrades
- used to prevent harmful proteins from being translated
CRISPR-Cas9 genome editing
- endogenous
- crDNA pairs with tracrRNA and Cas, which then scans DNA and cuts sites matching the crRNA spacer and flanked by a short CAS9-recognition motif, defending from phage DNA
- for editing, Cas9 creates a double-strand break at the target, then repairs by NHEJ or by HDR if a donor template is provided
Reporter gene
A gene whose expression is easy to assay phenotypically, and gets attached to a gene’s promoter to see when it is active
Transcriptional fusion
tells you where/when a protein is on
Translational fusion
tells you where/when a protein is on + where the protein goes
