- Plasmids
Plasmid Vectors
T-vector is used for cloning for PCR products- has an overhang on PCR product. It induces competency in bacteria cells which uptake the pasmid by transformation. DNA and plasmids are digested with the same enzyme and DNA is added to he digested plasmid and DNA ligase is added. Plasmids are inserted into the cell and the cells are grown in the presence of the selection agent. The plasmid then carries the gene for antibiotic resistance. Selectable markers in plasmid such as ampicillin resistance to selective transformants.
Sequencing
Plasmid vectors allow specific DNA to be rapidly grown up in large numbers, plasmids can then be re-isolated from teh bacteria. Vectors commonly used as a high quality template for DNA sequencing.
Recombinant Expression of Genes
occurs in prokaryotes and eukaryotes (often yeasts) however eukaryotic genes are often not processed correctly in prokaryotic expression systems and introns are not removed so post translation modification may be incorrect.
Gene Therapy
This is the insertion of a working copy of a gene into cells to treat disease as it repairs the faulty gene and expresses something which will be beneficial with respect to a disease. Delivery challenges include viruses, stem cell and coated nanoparticles. Triggering oncogenes is only used in cases where there is no other hope.
RNAI
this is RNA interference and is responsible for gene silencing/knockdown and uses small pieces of double stranded RNA and suppresses the expression of genes with sequence homology. Research goes into understanding the function of genes by silencing them. Interfering RNA is a natural system in eukaryotesand deals with RNA viruses and prevents expression of specific proteins by destroying specific mRNA and is triggered by the presence of dsRNA longer than 20bp (base pairs) and transmits from cell to cell.
SyntheticiRNA is a small molecule inhibitor of any gene of choice and can be seen in neurodegenerative disease and also used for treating acute infections, silencing proinflammatory genes to prevent tissue damage and controlling cancer by silencing regulators involved or oncogenes.
Vaccines
these can be live attenuated or DNA. The live attenuated vaccines manipulate the disease causing organism and have virulence genes removed. The DNA vaccnie uses vector/subunit vaccins. If the DNA is taken up by an animal cell and transcribed and translated-you get immune response and immunisation.
Learn More
Bacterial DNA: The Role of Plasmids
Scitable: Plasmids
Mayo Clinic: Gene Therapy
Questions
- what is a plasmid?
- describe a live attenuated vaccine and a DNA vaccine
- How does gene therapy work?
- what is RNAI and what is its function?