To detect the DNA or RNA of interest in the cell.
Northern blots are used to identify RNA – don’t localize or tell where in the cell a protein is.
Same procedure as southern blot, except using different buffers, and lets you see RNA
Southern blots are used to identify DNA - don’t localize or tell where in the cell a protein is.
Take DNA in cell, denature, expose to radioactive probes that attach to DNA that has been transferred onto filter. Probes only target the sequences you are interested in. Rinse excess radioactivity/fluorescence.
(fluorescence in situ hybridization) is a cytogenetic technique used to detect and localize the presence or absence of specific DNA sequences on chromosomes. FISH uses fluorescent probes that bind to only those parts of the chromosome with which they show a high degree of sequence similarity. Fluorescence microscopy can be used to find out where the fluorescent probe bound to the chromosomes are. FISH is often used for finding specific features in DNA for use in genetic counseling, medicine, and species identification. FISH can also be used to detect and localize specific mRNAs within tissue samples. In this context, it can help define the spatial-temporal patterns of gene expression within cells and tissues.
Whether genes are upregulated or down regulated in certain conditions.
Takes advantages of chips that are generated where you have multiple single stranded copies of genes with themes (growth factors, cytokines etc)
Take samples and isolate mRNA then convert to cDNA and label with fluorescent probe. Hybridize to microarray. Where are the green/red/yellow spots? The key for the chips tells which single stranded DNA that is or which gene is that that has been bound to a cDNA construct from sample XYZ. Targets differences between two samples or in certain conditions.
Antibodies – tag so you don’t need to generate an expesivie antibody for protein
Purifies protein rapidly, tags separate on column.
Make sure tag isn’t doing anything to cell function
How to use proteins, that are themselves fluorescent, to identify proteins of interests
GFP is a tag that has a construct that has an inducible promoter and has several sites for restriction endonucleases to insert DNA of interest. The construct won’t be expressed until you add the sugar that activates it. ITPG sugar activates lac promoter and the lac promoter begins to turn on transcription and translation of the protein plus GFP.
You can use living cells with GFP and fluorescent microscopy.
We can insert a gene of interest and we have a gene that confers drug resistance to those cells that are transfected with the plasmid. Not all cells stably transfect into genome. Those that have, will have ampicilin resistance. After a while, only those that have stably incorporated live. All others die off.
Knockout to remove active gene – difficult in mammalian, so you use embryonic stem cells injected into target gene replaced by mutation to generate progeny in a pregnant mouse that now expresses mutant/altered gene. Some have the mutation. Mate mice with normal mice so in F2, look for males and females with copies of mutated genes. Can be in somatic cells. Transgenic mice are looked at for the function of that gene.
Double stranded RNA has been known to be considered viral.
Double stranded RNA will be targeted by dicer, which cleaves the ends of the double stranded RNA making an siRNA (small interference RNA). The presence of siRNA is an indicator of these mechanisms of infection.
RISC proteins attach to siRNAs and degrade 1 strand of siRNA leaving a single strand which can:
Bind to message that has been generated which inhibits translation
Enter nucleus and bind complementary DNA to inhibit transcription.