Chapter 9 - Biotechnology Flashcards
Restriction Endonuclease
Enzyme which cuts DNA at a particular base sequence.
100’s of unique restriction enzymes exist.
2 ways restriction enzymes cut DNA & explanation
Sticky ends: Restriction enzyme leaves overhanging ends on DNA strand leaving a base sequence exposed without complementary base.
Blunt Ends: Restriction enzyme leaves straight ends
Polymerase Chain Reaction (PCR) steps;
1.) Denaturation - 96°:
DNA strands separate as hydrogen bonds are broken.
2.) Annealing - 68°:
Priming sequences (primers) are added to 3’ end of original strand.
3.) Elongation - 72°:
DNA polymerase binds to primer and synthesises remainder of DNA strand in direction 5’ to 3’.
4.) Repeat multiple time (25 usually)
Gel Electrophoresis explanation:
- Single type of restriction enzyme cuts up DNA samples into defined lengths.
- DNA samples are injected into wells within agarose gel.
- An electric current is sent through the gel sending the DNA fragments towards the positive electrode at he opposite side.
- DNA post electrophoresis can be revealed by staining the gel
DNA Markers (Gel Electrophoresis):
A mixture of DNA molecules with known molecular weights are ran simultaneously in order to estimate DNA molecule size of unknown sample.
Southern blotting (Gel Electrophoresis):
Technique which transfers DNA onto nitrocellulose membrane. Dry absorbant paper draws up moisture and dye, the DNA sticks to nitrocellulose sandwiched between the gel and absorbant paper.
Gel Probe ((Gel Electrophoresis):
Single DNA or RNA )containing radioactive tag or fluorescent dye) that binds to DNA under investigation
What net charge does DNA have?
Negative. Due to negatively charged phosphates in the DNA’s backbone.
Recombinant plasmids steps:
- ) Both plasmid & DNA fragments are cut with the same restriction enzymes
- ) Hybridise DNA - Fragments of matching sticky ends form hydrogen bonds with complementary base pairs.
- ) Ligation: DNA ligase used to join sugar phosphate backbones.
- ) Transformation: Modified plasmid re-inserted into bacteria cell by: CaCl, Heat, Electric shock
- ) Isolation and cloning of transformed bacteria. Isolated through antibiotic resistance / fluorescent gene
Use of calcium chloride and heat treatment in bacterial transformation.
CaCl dissociates into Ca+ ions which are attracted to negative charge of plasmid and phospholipid cancelling out their charge and increasing permeability.
Rapid heat treatment pushes plasmid into cell.
DNA Profiling:
What & how:
DNA profiling compares individuals based on the different lengths of repeats such as STR & VNTR
STR: Repeat sequence length 2-5 bases
VNTR: Repeat sequence length 5 bases
- Number of repeats different for each person.
- Use gene probes to find repeats
Somatic gene therapies
- An altered lab virus, unable to reproduce, and containing gene of interest is sent to infect patients specific cells
- Genetically altered cells producing the desired protein or hormone.
- Viral vector can infect cells directly in the body or can infect cells removed and cultured in a lab before being returned to the body.
Germ-line Therapy
Insertion of genes into germ line cells to change genetic make up of offspring.
Future generations effected
Totipotent vs Pluripotent vs Multipotent
Totipotent: Can give rise to all cells in body
- can give rise to entire organism
- Must be acquired before blastocyst stage (approx 8 cell embryo)
Pluripotent; -Can give rise to all cells in body
- Cannot give rise to entire organism
Multipotent: gives rise to limited range of cells within tissues.
Embryo Splitting
- Natural embryo splitting results in twins
Artificially:
-Embryo coating (which stimulates cell division is removed) - Embryo split artificially
- Two embryo’s (containing totipotent cells) are implanted into surrogate mother.
- Two offspring are identical
Somatic Nuclear Transfer
- nucleus removed from somatic cell (a) is added to an annucleated egg (b) cell through electric shock treatment.
- Egg os now 2n and is stimulated to divide into an embryo.
- Offspring has chromosomal DNA of (a) and chromosomal DNA from (b).
Adult stem cells
+ advantages & Disadvanatges
- multipotent
- found in organs and tissues
Ad; Does not require embryo
DisAd: difficult to isolate in cell
contain self antigens (rejection)
less versatile
Steps of stem cell differentiation and therapy
- ) Stem cells taken from embryo or adult tissue and are cultured
- ) Differentiation factor is added to stem cells to trigger them to divide into different cell types
- ) News cells are surgically implanted to replace damaged and diseased cells
Liposome vector:
Ad & Disad
Single membrane phospholipid membrane which fuses with cellular membrane.
Ad: Contains no viral DNA
- Can be made to target specific cells by adding receptor molecules
Disad: Not efficient at transferring genes into cells
Viral Vector:
Ad & Disad
Gene inserted into deactivated virus which is used to inject DNA into the cell;
Ad; Have the ability to integrate DNA into the cells chromosomes (or not)
Disad: Immune system attacks the virus
Plasmid Vector:
Ad & Disad
- injection of a recombinant plasmid into a cell
Ad: No viral or bacterial genes- Immune system does not mount a response
Disad: DNA not stable in this form.
- Immune system does not mount a response
What is bacterial transformation? And what is its purpose
Recombinant plasmid (containing specific gene) is inserted into the bacteria through heat shock treatment and CaCl. Purpose is to produce large quantities of the desired protein.
Is Biotechnology is natural? argyements
Yes: Genetic modification occurs through selective breeding which has existed for years
No: Biotechnology different to selective breeding as biotech involves the transferring of genes between species
Effects Biotechnology has on the envrionment:
Good & Bad
Good: - Little gene transfer occurs
- Reduce use of toxic herbicide and pest control
Bad: Herbicide resistant crops could encourage farmers to use more herbicide
- Higher gene transfer may occur between closely related species, therefore weeds could become resistant also
- It may not be known what transgenic organisms escape into the environment
GMO’s For & against;
For: Strict guidelines to ensure GMO’s are safe
increase durability of food
Reduce wastage of water, fertiliser
Against: Long term effects unkown
