Molecular Biology Techniques Flashcards
What is the central dogma of gene expression?
DNA –> RNA –> protein
What are 3 ways to analyse gene expression?
- RNA expression
- Protein expression & localisation
- Analysis of molecular interaction
What is does a hybridisation method include?
One strand of DNA & one strand of RNA
What is a probe?
DNA sequence that is complimentary to the RNA sequence you want to analyse - labelled in some way that we can analyse.
What are 2 ways of analysing RNA expression?
- Northern blot
- Microarray
Explain the steps of Northern blotting
- RNA separated by electrophoresis - will see major bands of rRNA
- RNAs are transferred to a membrane
- Probe - complementary to your chosen sequence - labelled.
Explain the steps involved of microarrays
- Oligonucleotides (short DNA molecules) attached to a spot in a chip.
- Each spot has a different oligonucleotide corresponding to a specific gene.
- Fluorescent cDNA is made from RNA
- Fluorescent cDNA is applied to the chip and allowed to hybridise.
How does microarrays measure relative mRNA levels?
Lots of fluorescence would signify that lots of cDNA has bound to the oligonucleotides. As a result, this would mean that there was lots of mRNA
How do we compare levels of transcription using microarrays?
Analysis of the colours produced via fluorescence will signify how much cDNA has been produced by each source
What is the technique used to analyse RNA localisation?
Fluorescent in situ hybridisation
Describe the steps of FISH (fluorescence in situ hybridisation)
Probe is labelled with a fluorescent marker & visualised using microscopy. Different colours will fluoresce in different locations, depending on where they have bound to.
How can reporter genes be useful?
If reporter genes are cloned next to the promoter region of a gene of interest, expression of the reporter gene (which may fluoresce) can signify the expression of the gene of interest.
- as a result, high level of fluorescence = high level of gene expression.
What are 2 uses of reporter genes
- protein localisation
- to measure expression
What are 2 ways of analysing protein expression & localisation?
- using protein-specific antibodies
- fusion proteins & reporter genes
How do protein-specific antibodies work?
- The primary protein-specific antibody attached to the protein. - - - The secondary antibody attaches to the primary antibody.
- The secondary antibody is usually conjugated to a molecule that allows detection
Describe the steps of Western blotting
- Proteins separated by electrophoresis
- It is then transferred to a membrane
- Detection is using a protein-specific antibody and a labelled secondary antibody
What type of detection molecule is usually conjugated to a secondary antibody?
A fluorescent molecule
What is an example of fluorescent protein?
GFP (green fluorescent protein)
How can fusion of the fluorescent protein (GFP) bound with a gene of a protein of interest be useful?
Expression of the protein will lead to fluorescence and therefore detection
What is the use of a pull-down assay?
A way of analysing protein interaction in vitro
What is an organism that can be used to make a lot of recombinant DNA?
E. coli
What is the process of a pull-down assay?
- Make a cell lysate
- Bind GST (Glutathione S-Transferase) to the affinity ligand.
- Wash away any unwanted stuff (purified recombinant protein)
- Investigate what can bind to protein
Explain the process of immunoprecipitation
- Use antibiotics to directly bind the preferred protein.
- See what interacts with it (co-immunoprecipitation)
What are the disadvantages of immunoprecipitation?
- Antibodies aren’t easy to make (expensive, not readily available, often in limited supply)
What is a yeast two-hybrid?
The fusion of proteins. This occurs when transcription is activated. This is facilitated as ‘bait’ binds to the DNA binding domain attached to the promoter, as well as the prey being bound to the transcription activator.
What is the use of chromatin immunoprecipitation (ChIP)?
Used to study interactions of proteins with DNA. Antibodies are then used to purify the protein.
What is the first step of chromatin immunoprecipitation?
Formation of a cross-link DNA-protein, in order for the interaction to survive harsh purification conditions.
