Topic 7.3 Gene Technology Flashcards
Gene technology
- The manipulation of genes in living organisms.
- Genes from one organism may be inserted into another.
Recombinant DNA
- DNA that has been formed artificially by combining DNA from different organisms.
(Marker genes are used to identify recombinant cells).
The first step in Recombinant DNA
- Isolating the DNA/gene of interest.
- Detergent is used to isolate the DNA from a sample.
- It breaks down the membrane.
(Protein may need to be removed using digestive enzymes). - There are three ways to isolate the gene:
1) Using reverse transcriptase
2) Using restriction endonuclease
3) The Gene Machine
Isolating gene: Using reverse transcriptase
- RNA is taken from a cell that produces the required protein.
- Reverse transcriptase causes a reaction in which complimentary DNA (cDNA) is made from mRNA and DNA nucleotides.
- The result is a single strand of cDNA.
—> DNA polymerase and free nucleotides are used to produce a double strand of cDNA
Isolating gene: Using restriction endonuclease
- Gene can be removed from the chromosome using restriction endonuclease.
- Different enzymes cut the DNA at a different positions.
- This is called a recognition sequence.
(Restriction enzymes are made by bacteria).
Isolating gene: Gene Machine
- Scientists examine amino acid sequencing in desired protein.
- Creates small non overlapping section of DNA which are joined to create the desired gene.
- PCR is used to amplify this copy.
Gene engineering
1) Isolating required gene (using restriction endonuclease).
2) Inserting the gene in a ‘vector’ (using ligase).
3) Transformation- the gene is delivered into the required cell for protein growth (using microinjection).
4) Identification of host cells that have taken up the gene.
5) Grow cells with the new gene on a large scale (cloning).
Transformation
- The vector needs to be placed back into the host cells.
1) Place the host cells in ice cold calcium chloride solution to make cell walls more permeable.
2) The plasmids are then added to the mixture is heat shocked (42°C for 2 minutes) which encourages the cells to take up the plasmids.
Transformation: Gene guns
- A method for the physical introduction of DNA into plant cells containing cell walls:
-DNA is shot into the cell at a high speed.
(Microscopic gold pellets are coated with copies of the DNA fragment).
Transformation: Liposome wrapping
- The gene to be inserted is wrapped in liposomes (spheres formed from a lipid bilayer).
- These fuse with the cell membrane and can pass through it to deliver the DNA.
Transformation: Microinjection
Injecting DNA directly into cells (often used in animals).
Transformation outcomes
- Successful transformation (gene is in plasmid)- 5% likely.
- Unsuccessful transformation (gene is outside the plasmid).
- No plasmid.
How do we make GM plants?
- Usually use the bacterium Agrobacterium tumerfacians which causes tumors in plants.
- Agrobacterium contains Ti plasmid, which transfers genetic material into plants.
- GM plant tissue can then be grown by tissue culture (as plant cells remain totipotent).
- Plants are cloned.
GM Soya beans
- They have been made resistant to herbicides.
- Fatty acid balance.
Knockout mice
- A lab mouse in which one or more genes have been turned off or ‘knocked out’.
—> To create a knockout mouse, scientists genetically engineer the animal by disrupting a gene of interest.
