Tutorial 5-7 Flashcards
The steady state transcript abundance is the result of?
The balance between transcription rate (mRNA production) and the mRNA degradation rate (mRNA destruction)
What experiments can be run to measure the abundance of mRNA but not the rate of transcription initiation.
- Norther blotting
- Microarrays
- RT-qPCR
What experiments can be run to measure the rate of transcription.
- Pulse labelling
2. Nuclear run-on assays
Pulse labelling principles
- Transcription rate is monitored in vivo by measuring the amount of radioactively labelled UTP incorporated by RNA polymerase.
- Transcripts of interest are detected by hybridisation to unlabelled cDNA.
How to interpret the pulse labelling results .
- Signals with higher intensity (darker spots) indicate that more mRNA molecules have be transcribed during the labelling period.
- Results give an indication of the relative rate of transcription, or stability / degradation (half-life) of transcripts over time.
Steps of pulse labelling assay.
- Radioactive nucleotide are inserted into the cell.
- The radioactive nucleotides are incorporated into nascent RNA chains.
- Chase period with all unlabelled NTPs, then isolate total mRNA.
- To identify mRNA of interest, the mRNA are hybridised to DNA of interest.
- Detect signals quantify and compare results.
Pulse labelling limitations
- The radioactive nucleotides we insert are competing with the nucleotides already present in the cells cytoplasm resulting in the low signal.
- For genes with low transcription rates, prolonged pulse labelling may be needed for detection, this is a problem because the radioactive nucleotides can cause degradation of the transcripts.
What is pulse labelling used to determine.
The half-life of mRNA
Nuclear run on assay
- Radioactive nucleotides are incorporated into a nuclei that has been isolated from cells(in vitro)
- RNA polymerase molecules become “frozen” on genes in the isolated nuclei.
Why is nuclear run-on assay favoured over pulse labelling
- Degradation takes place in the cytoplasm but the nuclear run-on assay is performed within isolated nuclei(in vitro)
- Short labelling time (10-15 min) this leaves little time for degradation.
- Can detect low abundance of mRNA
Advantages of nuclear run on assay.
- Results are not influenced by mRNA degradation.
2. Mor radioactive nucleotides are incorporated because cytoplasm is removed.
Bisulfide sequencing is used for?
Detecting DNA methylation
How does bisulfides sequencing work?
- Unmethylated cytosine is converted to uracil by the bisulfite- treatment. Since uracil is not a nucleotide used in DNA this will be repaired to Thymine.
- Methylated C are not changed by bisulfite treatment thus remain C’s.
.3. When a untreated sequence is compared to the bisulfide sequence. If both sequences have a C present at a position that C is methylated, if the untreated seq has a c at a position but the treated seq has a T at a position that is a unmethylated C.
What are DNAase 1 hypersensitive sites.
- Nucleosome- free promoter regions
2. Nucleosome free enhancer region
What are DNase 1 insensitive sites
30nm or higher packed chromatin
What DNase 1 sensitive sites
Beads on a string also called 10nm packed chromatin
Techniques to study chromatin structure
- DNAse 1
2. MNase
Simplified steps of DNase 1 and MNase digestion.
- Lyse cell
- Extract nuclei
- Permeabilise nuclei
- Digest with DNase 1 or MNase
- Quench
- Sequence digested DNA
How interpret DNase 1 results
- More open chromatin structure will be digested at Lower DNase 1 concentrations.
- Therefore the higher the DNase 1 concentration needed the tighter the chromatin is packed.
- From this we can tell if the genes belonging to the chromatin are transcriptionally active. Open chromatin= active. Closed chromatin = inactive
CDNA synthesis steps
- Oligo dT primer is bound to mRNA
- Reverse transcriptase copies first cDNA strand.
- Reverse transcriptase digests and displaces mRNA and copies second strand of cDNA
- Double stranded cDNA is created.
Steps of recombinant protein production
- Isolate mRNA
- Run a RT-qPCR
- Insert cDNA into expression plasmid.
- Purify protein
Components that go into expression plasmid.
- Mammalian promoter
- Cloned gene
- Selection gene
- Eukaryotic terminator (polyA signal)
- Antibiotic resistance (for bacterial)
- Bacterial origin of replication
- Eukaryotic origin of replication
Why do expression plasmid need a mammalian promoter.
To enable the transcription and translation of your gene of interest , there needs to be a eukaryotic promoter, Kodak and start site in front of this insert (GOI).
Why do expression plasmid need antibiotic resistance.
To enable selection between recombinant and non recombinant plasmids.
Steps of purifying proteins.
- Affinity ligand( attract our protein of interest) attached to a chromatography support and packed in a column.
- The ample from our plasmid is flowe through the column, allowing the target molecules to bind to the affinity ligand.
- The sample is washed to remove any unwanted proteins and molecules.
- Elution buffer is applied to release the protein of interest from the column, now producing a pure sample of our protein.
What are reporter genes used for
Reporter genes encode a protein that can easily be detected and quantified.
Examples of reporter genes
- CAT
- Bacterial LacZ gene
- Luciferase
- GFP
How is the reporter gene CAT detected?
Detect radio labelled product by auto radiography
How are the reporter genes bacterial LacZ genes detected?
Blue product when provided with X-gal substrate.
How are the reporter genes luciferase genes detected?
Fluorescent protein, detected with fluorometer.
How are the reporter genes GFP detected.
Fluorescent protein encoded by gene of a jelly fish, one of the most popular reporters, versatile and stable, fluoresces bright green when exposed to UV light, no substrate needed.
How do I conduct a promoter/reporter experiment ?
- Isolate promoter region (DNA)
- Clone into reporter plasmid vector.
- Transfers into cell line/ introduce into organism of interest.
- Do reporter assay.
What is the Promoter deletion analysis used for.
Used to determine what effect different regions of a promoter has on transcriptional initiation.
How do we do the promoter deletion analysis
- Make mutations of control region
: 5’ end deletion series
: internal deletions
: point mutations
Explain 5’ end deletion series
Incremental deletions of promoter from one side to yield smaller promoter fragment
Internal deletions
Delete small regions within promoter
Point mutations
Alter specific bases within promoter
How do you investigate whether a protein binds to a DNA control region.
Electrophoretic mobility shift assay
Principle of electrophoretic mobility shift assay
- If a short labelled DNA fragment representing a control element is bound by proteins, it will migrate slower through a gel than the naked DNA fragment.
- Protein-DNA complex will therefore cause a shift in location of probe.
Principle DNase 1 foot-printing
When a DNA fragment is bound by a specific protein, it will be resistant to digestion by DNAase 1.
DNase 1 Assay requirements
- End-labelled DNA fragment containing regulatory elements.
- Protein extract from relevant cells
- DNase 1 enzyme used at conditions where every DNA molecule will be digested once.
- Separation of DNA fragments by PAGE and visualisation by autoradiography.
Chromatin immunoprecipitation Principle.
Technique to precipitate a protein antigen out of solution using an antibody that specifically binds to that particular protein.
Application of protein immunoprecipitation principle.
To isolate and concentrate a particular protein from a sample containing many thousands of different proteins.
Steps in immunoprecipitation
- Suitable antibody is added
- Antibody binds to protein
- Protein A or G added to make antibody protein complexes insoluble. M
- Centrifugation of solution pellets antibody-protein complex. Removal of supernatant and washing.
Application of chromatin immunoprecipitation.
Identify transcription factor binding sites in DNA
Identify location of histone modification in DNA
Identify regions of DNA that is methylated.
In EMSA why when a specific competitors is added to the protein hot probe mixture will we only see one band ?
Because the specific competitor will compete with the Hot probe and instead of the protein’s binding to the Hoy probe they will bind to the specific competitors. The specific competitors are not radioactive therefore this will not be picked up on X-ray resulting in only one band (the Hot probe) when in reality there are two( hot probe and proteins bound to competitor)
Steps of EMSA
- Radioactively Label a nucleotide acid probe
- Isolate proteins or purify a particular TF
- Incubate at room temperature in an eppendorf
- Run the samples on agarose gel
- Expose X-ray film to gel
Why do we add a specific competitors to our test tube containing our proteins and hot probe.
To ensure the binding we are witnessing is due to specific binding not non specific binding, such as the bind of histones which can bind to almost all DNA
How do we tell in EMSA if the binding of a transcription factor is specific on non-specific using the nonspecific competitor.
- If it is specific it will not bind to the non-specific competitor and a band will show in that lane.
- If it is non-specific it will bind to the non-specific competitor and no band will show .
How do we tell in EMSA if the binding of a transcription factor is specific on non-specific using the specific competitor.
If it is specific it will bind to the specific competitor only leaving non-specific transcription factors to bind to the hot probe. Therefore if it is specific you will see no bad because the free non-specific TF have not bound. But if is non-specific you will see a band because the non-specific TF have bound to the hot probe .
Supershift
Retardation caused by an antibody.
In EMSA what is the moving up of the DNA on there gel called
Mobility shift
What type of gel is used in DNase1 footprinting and why?
Polyacrylamide gel because it has a much higher resolution to separate fragments that are very close together in size.
Why does sonification need to be done In ChIP assays
To break the chromatin into smaller pieces to:
- Make analysing it further easier
- The smaller pieces are soluble In solution and whole chromatin is not.
