Flashcards in Molecular Tools in Developmental Biology Deck (20):
We will be studying techniques that allow us to do two things:
1) Assess when and where a gene is expressed
2) Perturb gene expression or function
What is the easiest way to study a protein?
Work with the DNA that encodes it.
What is the difference between [DNA and tRNA] vs. [mRNA]?
mRNA does not have any introns.
How do you make cDNA?
You use reverse transcriptase to create a copy of mRNA. This is useful in studying a protein.
What is Nucleic Acid Hybridization useful for?
You can create strands that are complement to the ones that you want, in order to probe for them.
Describe in Situ Hybridization
You create a cDNA with a dioxygenin on it. This cDNA is complement to the mRNA you want to find. Finally, you can use an antibody as a probe that looks for the dioxygenin, and hence the mRNA.
Describe Indirect Immunostaining
You add primary antibodies that connect to ANY molecule of interest. You then can add fluorescent secondary antibodies to identify the molecule of interest.
Describe tagging a protein with GFP
You can add a piece of DNA that creates GFP so that when a molecule is created, it has a small fluorescent tail piece.
What is confocal microscopy useful for?
Eliminating out of focus fluorescence from thick specimens like embryos.
Over expression of mRNA tests for?
Removing the function of a gene or protein tests for?
What two removal methods acts at the level of the mRNA?
anti sense oligonucleotides, and RNAi
What removal method acts at the level of proteins?
morpholino antisense nucleotides
How does RNAi work?
Short strands of RNA create RISC complex that search and destroys normal mRNA. These short strands can either directly be siRNA's, or can be formed by introducing double stranded RNA.
How do morpholinos work?
These chemically modified oligonucleotides bind to and prevent translation of mRNA
What are the four types of modification at the DNA level?
Transgenics, homologous recombination, CRISPR/Cas9, and mutations.
What is it called when you add genetic material?
Describe the process of homologous recombination?
Create piece of DNA and insert at gene site via recombination.
Use double drug selection to identify ES cells with said DNA exchange.
Place these cells into chimeric embryo
Breed until you get homozygous knockouts.
What is CRISPR/Cas9 good for?
Replacing specific DNA