Lec. 27: Biotechnology Flashcards
Biotechnology
Manipulation of organisms or their compnents to make useful products
Recombinant DNA
nucletotide sequences coming from two different sources, often two species, are combined in vitro into the same DNA molecule
DNA cloning
To work directly with specific genes, scientists prepare well defined segments of DNA in identical copies
- yields multiple copies of a gene or other DNA segment
Why are bacterial plasmids used as cloning vectors
- readily obtained
- easily manipulated
- easily introcued into bacterial cells
- rapidly multipy once in bacteria
Plasmids
small circular DNA molecules that replicate from bacterial chromosome
How do you clone a gene?
- Gene inserted into plasmid (restriction enzyme cuts DNA at restriction sites and ligase binds them together)
- Plasmid put into bacterial cell (bacteria likes to take up plasmid)
- Host cell grown in culture to form a clone of MANY cells containing the cloned gene of interest
- Cloned genes can be used now for basic research and applications
Cloning vector
In gene cloning, the original plasmid is called the cloning vector.
A cloning vector is a DNA molecule that can carry foregin DNA into a host cell and replicate there
Bacterial defenses against phages
- Bacteria has developed their own immune system and have their own defense against phages
- Bacterium’s own DNA protected from restriction enzymes by being methylated in key locations
How do restriction enzymes make recombinant DNA?
How can DNA bind together?
- Restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites
- Restriction enzymes make many cuts making restriction fragments
- When enzymes cut DNA staggered, fragments with “sticky ends” are produced that bond with other sticky ended fragments. It is then sealed by DNA ligase
CRISPR
acronym, what does it contain and what is its function, structure
Clustered Regularly Interspaced Short Palindromic Repeat
- Contain snippets of DNA from viruses that have attacked bacterium
- used by bacterium to detect and destroy DNA from further attacks by similar viruses
- Palandromic repeat followed by a spacer sequence in a pattern
Cas9
What does genome editing require?
CRISPR ASSOCIATED PROTEIN 9
- Cas9 is a RNA guided DNA nuclease for direct cutting of a DNA sequence which can be repaired with a custom sequence
Genome editing requires a Cas9 protein, guide RNA, and a repair RNA template (replacement sequence)
How can an animal be cloned? How does it work?
NUCLEAR TRANSPLANTATION
(Need mammary cell donor cells and egg cell donor)
1. Culture mamary cells
2. Take egg cell from ovary and remove nucleus
3. Fuse cultured cell and empty egg and nucleus forms inside empty egg
4. New cell is grown in culture and development of embryo begins
5. Embryo implanted in 3rd lamb
6. New identical lamb is genetically identical to mammary cell donor
What part of Dolly does not come from the mammary cell donor
The mitochondrial DNA comes from the mother
What are problems associated with animal cloning?
- only a small percentage of cloned embryos have developed normally to birth, many defects
- Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor in order for genes to be expressed appropriately
Stem Cell
A relatively unspecialized cell that can reproduce itself indefinitely and differentiate into specialized cells of one or more types
