Section 8 - In-vivo and In-vitro cloning etc. (until end) Flashcards
What does in-vivo mean?
In life
What does in-vitro mean?
In glass
What is the importance of sticky ends?
DNA from different sources can be joined together if they have the same sticky end
How can sticky ends be joined together?
Using DNA ligase
Joins the sugar-phosphate backbone together
What is the new DNA molecule combing sticky ends and DNA called?
Recombinant DNA
What is a plamid?
Used to transport DNA into a host cell
What are plasmids?
Most commonly used vector
Why are plasmids useful?
Nearly always contain antibiotic resistant genes
What is the role of the plasmid in in-vivo cloning?
Contains 2 antibiotic resistance genes
One is disrupted when restriction enzymes cut open the plasmid
Other used to select cells which have taken up the plasmids
What is the process of in-vivo cloning?
- DNA is cut using restriction enzyme
- Same restriction enzyme is used to cut open the plasmid
- Enzyme is denatured
- Plasmid now has sticky ends complementary to donor DNA
- DNA ligase joins 2 DNAs together
- Recombinant DNA is now created
What is transformation in in-vivo cloning?
Insertion of DNA into host cell
Plasmids must be reintroduced into host cell
Bacteria, plasmids and Ca2+ ions are mixed together
How is a bacteria containing the plasmid identified?
Growing bacteria in a medium that contains an antibiotic
Found in the plasmid and bacteria will only survive in the presence of an antibiotic if the genes are present and functioning
How is bacteria containing plasmid with DNA fragments identified?
Gene markers are used to identify which plasmids have taken up the DNA fragment
Gene marker is disrupted if the DNA fragment is present
What can gene markers be?
Resistant to an antibiotic
Fluorescent protein
Enzyme whose action can be identified
Why is replica plating used?
Used to identify the bacteria with the recombinant plasmid
Bacteria are transferred to identical positions on plates containing ampicillin and then tetracycline
Bacteria with the donor DN will be able to grow on ampicillin, but not tetracycline
Why are gene markers necessary during in vivo gene cloning?
Gene markers are used to identify which bacterial plasmids have taken up DNA fragments
What is an advantage of using fluorescent gene markers rather than antibiotic gene markers?
Not killing bacteria you want
Easy to identify
What are some of the arguments for and against the use of DNA technologies?
For:
Microorganisms modified to produce large range of substances –> used to treat diseases
GM plants crops can help to prevent certain diseases
GM crops can be engineered to have financial and environmental advantages
Against:
Impossible to predict accuracy
GM bacteria often have antibiotic resistance marker genes that have been added
Long-term consequences cannot be identified
Ethical considerations
What is transfection?
Inserting the correct gene into a cell
Only suitable for disease caused by a single gene
What is somatic (body cell) cell therapy?
Copies of the correct gene are inserted directly into the somatic of the sufferers
Doesn’t prevent the disease from occurring in the next generation
Has to be repeated many times as the effects don’t last very long