functional genomics 2 Flashcards
what is genomics?
The study of genes and their functions.
What is the genome
The genome is the entire DNA content that is
present within one cell of an organism
What does functional genomics use?
uses genomic data to study gene and protein expression and function on a global (genome-wide) scale
What is the focus of study in functional genomics?
uses genomic data to study gene and protein expression and function on a global (genome-wide) scale
What is transcriptomics?
the whole complement of transcripts in a tissue, organ, or organ system.
What is proteomics?
the whole complement of proteins in a tissue, organ, or organ system.
Function of large scale, high-throughout assays?
track many genes or proteins in parallel under different experimental or environmental conditions (e.g. withsamples from patients and healthy individuals)
Whats the genome wide approach?
“genome-wide” approach allows the function of different parts of the genome to be discovered by combining information from genes, transcripts and proteins
What techniques are used in genome wide approach?
Assays to measure changes insynaptic plasticity performed
alongside assays that quantifychanges in proteins involved in a range of pathways e.g.
MAP kinase signalling cascade
Functional genomics integrates information from various molecular methodologies to …
…gain an understanding of how DNA sequence is translated into complex information in a cell
(DNA → RNA → Proteins → biological process)
slide 6
Proteins can be separated according to…
… their molecular weight
electrophoresis is usually conducted in…
… a vertical electrophoresis system.
the matrix where proteins are separated according to their molecular weight is…
… polyacrylamide instead of agarose but the principle is the same.
in gel electrophoresis, the smaller the molecule, the…
…faster they will migrate.
DNA molecules are uniformly charged. What does this mean?
the sugar phosphate backbone is negatively charged regardless of DNA sequence.
DNA molecules are uniformly charged, so…
… migration is solely due to molecular weight.
Protein molecules consist of …
different amino acids that may have different charges
charge of DNA molecules?
negative because of phosphates.
Why are the charge differences between amino acids important?
The charge differences between amino acids are important in determining protein structure and function but the presence of different amino acids means that proteins do not have uniform charge
Proteins are denatured prior to…
electrophoresis
Proteins are denatured prior to electrophoresis by addition of …
…mercaptoethanol and SDS (sodium dodecyl sulphate)
Mercaptoethanol disrupts …
disulphide bridges between cysteine amino acid pairs by reducing the double bond - SDS binds all over the protein negating its charge
As SDS is negatively charged the presence of excess SDS molecules binding to protein results in …
…a uniform negative electric charge on protein molecules
MW = ?
= molecular weight marker
Protein gels can be…
…blotted
the principle of blotting in protein gels is the same as…
… DNA and RNA gels.
Describe the process of protein gel blotting.
Proteins and buffer are forced to migrate through filter paper but proteins are unable to travel through a membrane (PVDF – polyvinylidine fluoride)
This produces a duplicate of the gel on a membrane
How is blotting accomplished in protein gels?
The difference is that this is accomplished by electroblotting rather than capillary action
In the case of Southern and Northern blots we use ….
DNA probes to detect the presence of homologous sequences of nucleic acids
How do we detect proteins?
Using antibodies.
proteins can be bound by…
… antibodies
The vertebrate immune system relies on antibodies to bind to …
… antigens
The vertebrate immune system relies on antibodies to bind to antigens – these complexes are then recognised by…
…components of the immune system where they activate an immune response.
If we can bind protein with antibodies and label antibodies (as we label a DNA probe) then…
… we have a means of detecting specific proteins in a Western blot.
We can make use of the vertebrate immune system to …
… generate antibodies
The first step in creating an antibody that specifically recognises our protein is to …
… express the protein (creating the antigen)
Simple cloning techniques allow us to …
… insert a gene we want to express into a plasmid
If it is downstream of a strong promoter recognised by E. coli then if we transform E. coli with the plasmid…
… the protein will be produced.
The protein we want antibodies generated against is…
… purified and injected into an animal (usually a rat or rabbit)
The animal then …
… makes antibodies specific to that protein as it recognises the protein as foreign – after several weeks blood is harvested and serum produced.
slide 14
By producing antibodies specific to our protein we have solved one problem …
… –we have something that will bind to our protein
By producing antibodies specific to our protein we have solved one problem – we have something that will bind to our protein – but we still have another which is…
how do we detect binding?
The answer lies in using antibodies from …
another animal that are raised to the first antibody (if we inject IgG from a rabbit into a goat, the goat will generate antibodies to rabbit IgG)…
The answer lies in using antibodies from another animal that are raised to the first antibody (if we inject IgG from a rabbit into a goat, the goat will generate antibodies to rabbit IgG)
If we then collect those antibodies and covalently link another protein on to it that will give us a…
colour reaction then we can detect the first antibody.
As with a northern blot, spatial expression of the protein can be…
… studied.
Protein is extracted from…
…various tissues.
how is protein extracted?
this is quite a simple procedure just relying on gently lysing the cells and collecting the protein extract
The protein extracts are separated by…
…SDS-PAGE and then blotted.
As with a northern blot, spatial expression of the protein can be studied
Protein is extracted from various tissues (this is quite a simple procedure just relying on gently lysing the cells and collecting the protein extract)
The protein extracts are separated by SDS-PAGE and blotted
The western blot is then probed with …
… primary antibodies and finally with secondary antibodies with an enzymatic tag.
slide 15
In ELISA assays, the same principle as western blotting can be used to …
… make detecting protein quantitative
Antigen is bound to a surface and then primary antibodies, secondary antibodies, and finally substrate are
… added to detect antigen.
The same drawbacks for Northern blotting occur with Western blotting and ELISA –
it does not say in which cells the protein is expressed and is a ‘rough estimate’ of what is actually going on.
In the same way that the presence of RNA can be detected at the cellular level by the use of RNA in situ hybridisation we can examine…
… the cellular distribution of proteins
Sections can be taken of an organism and these can be probed with antibodies
Proteins are targeted to specific parts of the cell and it is important in our investigation of gene function to find the …
… subcellular as well as the cellular location of a protein.
slide 17
The subcellular localisation of proteins can also be
analysed using electron microscopy
Rather than relying on enzymatic reactions to detect antibody in this procedure
small gold particles (10nm) are attached to the secondary antibody and the presence of antibody is seen because gold is ‘opaque’ to the electron microscope
The big problem with analysing sections using immunohistochemistry is that …
… the cells are dead – remember that the cell is a dynamic environment, proteins don’t just stay in the same place necessarily
The big problem with analysing sections using immunohistochemistry is that the cells are dead – remember that the cell is a dynamic environment, proteins don’t just stay in the same place necessarily
In order to analyse what our proteins might be doing in the cell…
…we can make use of GFP
Protein-GFP fusions can be constructed as for …
… promoter-fusions – as long as you can construct this then you can transfect into cells.
go look at slide 19
By re-engineering the GFP protein it has been possible to…
… produce proteins that emit light at different wavelengths – red RFP, yellow YFP and blue CFP
