Lecture 27: Cloning Flashcards
What is the purpose of cloning in genetic engineering?
To put DNA of interest into a plasmid for the production of large quantities of DNA, RNA, or protein
Cloning can be used in the lab or industry.
What are the four essentials required for cloning?
- Bacteria ability to be transformed
- Plasmids
- Restriction enzymes
- Ligase
What do restriction enzymes do?
Cuts specific recognition sequences, typically 4-8 bp long
Give examples of sticky end and blunt end cutters.
- Sticky end cutters: EcoRI
- Blunt end cutters: HaeIII
What is the role of DNA ligase in cloning?
Reforms phosphodiester bonds broken by restriction enzymes
Ligase is essential for forming recombinant DNA molecules.
What is the process to make recombinant DNA molecules?
- Isolate gene of interest using RE
- Digest plasmid with the same RE
- Add digested gene, plasmid, and ligase together
- Form recombinant plasmids
How are competent bacteria prepared?
By heat and freeze treatment to make membranes leaky
What do cloning vectors typically contain?
- Antibiotic resistance genes
- Selectable markers (e.g., ampicillin resistance) * Promoters to express cloned genes
What type of vectors are needed for putting cloned genes into Eukaryotic cells?
Eukaryotic expression vectors
What is a gene gun and its primary use?
A device that shoots very small shots coated with DNA into cells, mostly used on plant cells
What are two methods of introducing DNA into Eukaryotic cells?
- Electroporation
- Chemicals that cause cell membranes to become leaky
What are some uses of cloned genes?
- Production of proteins for pharmaceutical use (e.g., insulin, interferon, TPA)
- Agriculture (e.g., transgenic plants like BT corn)
- Industrial uses (e.g., large scale production of enzymes)
What is BT corn and how is it beneficial?
Genetically modified corn that is toxic to certain insects due to a toxin gene from Bacillus thuringiensis
What potential problems are associated with recombinant organisms?
- Control of recombinant organisms
- Public perception
- Unexpected environmental effects
True or False: Cloned genes can be used to produce insulin.
True
Fill in the blank: The bacterium _______ produces crystal-like proteins that kill specific groups of insects.
Bacillus thuringiensis
What are ‘crippled organisms’ used for in labs?
To limit the survival of recombinant organisms outside the lab
