Genetic Strategies Flashcards
What is the forward genetic approach?
Seek to find the genes encoded by DNA that are responsible for a phenotype of interest
Summaries the forward genetic approach:
- Starts with phenotype
- A series of mutants with defects in a particular phenotype may be linked in the same molecular pathways/functions
- Enables the wild-type genes for this pathway to be identified and studied
Describe the forward genetic approach:
1) Isolate mutants with a phenotype of interest.
2) Define genes responsible for these phenotypes.
3) Clone/identify the genes.
4) Analyse the genes to predict encoded proteins
Describe the isolate mutants with a phenotype of interest stage of forwards genetics of forward genetics:
- Choose good organism for genetics
- Mutagenise (physical, chemical and biological mutagens)
- Screen for mutants with the desired phenotype (For essential cell functions require conditional lethal point mutants)
Examples of genetic model organisms:
- Saccharomyces cerevisiae
- Caenorhabditis elegans
- Drosophila melanogaster
What are physical mutagens?
- UV
- Ionising radiation
What are chemical mutagens?
- Base analogous
- Alkylating agents
What are biological mutagens?
Transposable elements
What are screen for the desired phenotype?
Temperature sensitive mutants (mutant protein is functional at the permissive temperature/non-functional at the non-permissive (restrictive) temperature)
Give an example forward genetics:
Cell cycle - fission yeast Schizosachhoromyces pombe
Describe define genes responsible for these phenotypes stage of forwards genetics:
Genetic complementation tests can determine if a phenotype arises from mutations in the same or separate genes
Describe clone/identify the genes stage of forwards genetics:
Clone genes by mutant rescue (mutant complementation)
Transform cells with gene/cDNA library
Isolate transform ants which rescue, or restore a wild type phenotype
What is a gene library?
Collection of recombinant clones
Describe analyse the genes to predict encoded proteins stage of forward genetics:
- Compare predicted protein sequence with database sequences
- BLAST = Basic Local Alignment Search Tool
- Gives clue to function, which can be testes
What is the eukaryotic cell cycle controlled by?
Protein phosphorylation
What is responsible for cell cycle control?
CDKs
How were CDKs discovered?
By genetic analysis
Which of the following sets of procedures is involved in forward genetics?
A) Alter gene in vitro; introduce into cell; determine phenotypic effects.
B) Clone gene into plasmid; hybridise mutant oligonucleotide; grow up in Escherichia coli; isolate desired mutagenised clone.
C) Isolate mutants; define genes responsible; clone the gene.
D) Transcription; RNA processing; translation.
C
What is reverse genetics approach?
Seeks to find the phenotypes linked to specific sequences of DNA
How does reverse genetics approach try and find out the phenotypes linked to specific DNA sequences?
- Complete knockout the function of a gene or alter its sequence
- Then observe the effect phenotypic affects
How is reverse genetics done traditional ?
Alteration to genes in complete vitro
How is reverse genetics done now?
Deleting, disrupting or producing mutations in a specific gene may reveal phenotypes that can reveal their functions
Summaries the mechanism to reverse genetics approach:
1) Alter gene in vitro
2) Introduce into cell
3) Determine phenotypic effects
Describe alter gene in vitro stage of reverse genetics?
- DNA sequences synthesised
- Use recombinant DNA techniques
Give an example of recombinant DNA techniques are used in reverse genetics:
Oligonucleotide-mediated site-directed mutagenesis
Describe Oligonucleotide-mediated site-directed mutagenesis:
- Gene in plasmid
- Denature and hybridise mutant oligo
- Grow up in E.coli
- Isolate desire clone
Describe introduce DNA into cell stage of reverse genetics:
Take up in cell: -Direct uptake of DNA or -Aid/Induce cell DNA uptake When take up: -transgene has transient expression and replicates on a plasmid -Chromosomal interaction (random or targeted to a particular locus) -Somatic or germ cell -Haploid or diploid
How is direct uptake of cell done in reverse genetics?
Incubate DNA with competent cells
Bacterial/yeast transformation or animal cell transfection
How is aided/induce cell DNA uptake in reverse genetics?
- Electroporation
- Microinjection
- Virus-mediated
- Ballistic (Gene gun/Cells with walls e.g. plants)
- Agrobacterium tumefaciens-mediated (Plants and some fungi)
Hoe are transgenic cells detected in reverse genetics?
- Selectable marker gene
- Yeast and bacteria often use auxotrophic mutants
Describe phenotypic effects:
Compare between controlled and experimented organism
How can homologous recombination be utilised for gene targeting?
- Breakage and rejoining of DNA
- Gene disruption/deletion
What may gene targeting need?
Marker genes or a marker recycling scheme
What does CRISPR stand for?
Clustered Regularly Interspaced Short Palindromic Repeats
Describe the steps of CRISPR-mediated viral defence in bacteria:
- Short viral DNA sequences is integrated into CRISPR locus
- RNA is transcribed from CRISPR locus, processed, and bound to Cas protein
- Small crRNA in complex with Cas seeks out and destroys viral sequences
How can CRISPR be used for gene editing?
- Double strand break
- Activation of gene
- Repressor of gene
What concerns are there about CRISPR and gene editing?
Ethical concerns in regards to humans
What can CRISPR be used with for gene editing?
gene insertion
What does CRISPR and gene insertion enhance?
Homologous recombination
What does targeted regulated expression of native genes mean?
Replacing native promoter for your favourite gene with and active one or regulatable promoter
What are promoter activity reporters?
Reporters identify when and where genes are switched on and off by the promoter
Examples of reports:
β-Galactosidase, Luciferase, Green fluorescent protein (GFP) reporters
When are reports a problem?
Problem for haploid organisms, problem with complex promoters, problem for organisms with no formal genetics
Why do we tag proteins with fluorescence?
Allows observation of where DNA is and what it is doing and get clues of their function
How can we avoid genomic editing?
By using RNAi
What does RNAi do?
-Cleavage of targeting RNA
or
-Translation repression and destruction of target RNA
or
-Formation of heterochromatin on DNA from which target RNA is being transcribed
What is RNA interference forwards or reverse genetics approach?
Reverse genetics approach
What is RNAi an experimental tool for?
Specific gene “knockdown”
What is high throughput?
Use of automated equipment to rapidly test thousands to millions of samples for biological activity at the model organism, cellular, pathway, or molecular level.
Why do we use high throughput?
-Organism are complex
