Reverse Genetics Flashcards
What is reverse genetics
Starts with the genotype, looking for the phenotype linked to specific sequences of DNA
Techniques of reverse genetics
-directed deletions and point mutations
-transgenesis
-gene silencing
Outline directed deletions and point mutations
Introduce specific, intentional and permanent changes to the DNA sequence
Gene knockout and gene knock-in
Outline transgenesis
Introduce a DNA segment from one organism into a different organism
Outline gene silencing
Gene knock down temporarily prevent the expression of a certain gene; generated using double stranded RNA- broken down by the cell
Cross over
Gene disruption- introduce circular homologous DNA, single crossover
Gene disruption- introduce linear homologous DNA, double crossover
Name the 3 steps of reverse genetics
Altering gene in vitro
Introduce into cell
Determine phenotypic effects
How to alter genes in vitro
Altering plasmids- multiple cloning sites, antibiotic resistance markers and an origin of replication
Carries foreign genetic material into another cell.
Recombinant DNA techniques
How to introduce vector to cell
-competent cells, bacteria take up DNA from their surroundings with heat
-electroporation, any cells electrocuted, membrane flips and takes up any DNA on the outside
-micro injection, inject DNA into eukaryote nucleus
-transduction, eukaryotes infected with an altered virus holding DNA
How to determine phenotypic effects
Separate out cells that did not take up the DNA using selectable markers like an antibiotic resistant gene
What is GFP (green fluorescent protein)
Isolated from jellyfish
First bioluminescent protein- glows under UV light
How does GFP work
Replace coding sequence with GFP
When the gene is expressed, GFP is expressed instead
Allows us to locate where a gene is expressed
Gal4 UAS system
Gal4 expressed instead of gene of interest
Gal4 binds to UAS-expresses GFP
Gal4 enhances expression of GFP
What is Cre
Recombination enzyme
What is Lox
Sequence in DNA that enzymes target
