Genetic manipulation - lecture 1 Flashcards
What is genetic manipulation?
Genetic manipulation is the formation of new combinations of heritable material by insertion of nucleic acid molecules, produces by means outside the cell, into a virus, bacterial plasmid or other vector system so to allow their incorporation into a host organism in which they do not naturally occur and can continue to propogate.
What are the three basic challenges in genetic manipulation?
1) purification of a sufficient amount of DNA
2) Manipulation of DNA in lab
3) Production of large amounts of DNA to study
How is DNA purified using alcohol?
Start by lysis of the DNA or RNA
2nd step is precipitation of DNA using alcohol and salt
- The salt neutralises the negative charges of the DNA molecule making it more stable and less water soluble, the alcohol (ethanol or isopropanol causes the DNA to precipitate out of the aqueous solution because it is not soluble in alcohol.
-The purified precipitate is then collected by centrifugation and suspended in a volume of choice
- When the DNA has been separated from the aqueous phase it can then be rinsed with alcohol to remove any unwanted remaining material and cellular debris. It is then usually re-dissolved in water for easy handling and storage
How can DNA be lysed?
Mechanical disruption (breaks open cell) using a tissue homogeniser, mortar and pestle, or by cutting the tissue into small pieces. Can also use detergents and enzymes (Proteinase K) or shake with beads
Why is mechanical disruption important for plant cells>
Because they have a tough cell wall
DNA purification using a collumn with DNA binding resin.
Cell is lysed releasing DNA into solution
Lysis solution is then put into a column in an eppendorf tube
Tube is centrifuged and liquid will go through binding material leaving only DNA
Add washing buffer and centrifuge again - this cleans DNA
Add elute buffer and put into clean centrifuge tube
DNA dissolved at bottom of tube in form of solution
What is restriction endonucleases?
Protein/ enzyme found in some bacteria that cleaves DNA at a specific site
How was Restriction endonucleases found to perform this task?
Some bacteria strains were more resistant to viral infections than others. They recognised invading phage DNA and cut it into small pieces.
Restriction enzyme then inhibited from cells and used in labs to manipulate DNA fragments
What are some features of restriction endonucleases?
- The most important restriction enzyme is “type II” initially isolated from Haemophilus influenza
- Each restriction enzyme recognises a specific short sequence of nucleotides (recognition site/sequence)
- Different bacteria species make different restriction enzymes
- Recognise a palindromic sequence and cleave within it
Who discovered restriction endonucleases and when?
Werner Arber, Daniel Nathans and Hamilton Smith in 1978
What are sticky and blunt ends?
Blunt ends are cut at the same position, sticky ends cut on opposite sides on the two strands
How are blunt ends connected?
Through ligase which repairs links/gaps creating phosphodiester bonds using energy ATP or NAD+)
What checks are address during quality control?
Check the recombinant plasmid cut the products again using the same restriction enzyme
Gel electrophoresis to separate and visualise the garments
- dye specific for nucleic acids added - ethidium bromide- Piet Borst
