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
