BIOTECHNOLOGY AND GENETIC MODIFICATION
1. Identify gene
2. Isolate gene
3. Amplify (increase concentration)
Gel Electrophoresis
- Used to separate DNA and protein according to size and charge.
- Gel is a porous medium.
- DNA is negatively charged.
1. Samples of DNA/protein are inserted into wells in a gel.
2. Gel is placed in a conducting fluid and a current is passed through it.
3. Molecules move through gel based on charge.
4. Smaller fragments/molecules move further through the pores in the gel while larger pieces don't travel as far.
- Placed in a water tank to act as a pH buffer; medium
- DNA samples are then dyed.
- Used in DNA profiling.
Polymerase Chain Reaction (PCR)
(Taq DNA polymerase + free nucleotides + DNA + DNA primers)
- Allows for process of DNA synthesis to happen inside a machine at a faster rate.
- Synthetic method to amplify specific DNA sequences (e.g. crime scene samples)
- Taq DNA polymerase is used for PCR at 72 degrees Celsius.
From heat resistant bacterium - "Thermus Aquaticus" - lives in hot springs
- Copies up to 1000 nucleotides per minute.
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1. DENATURATION - DNA sample heated to 95 degrees Celsius to break hydrogen bonds and separate strands.
2. ANNEALING - DNA sample cooled to 54 degrees Celsius, primers allowed to attach to opposite ends of target sequence.
3. ELONGATION - Taq DNA Polymerase copies the strands at 72 degrees Celsius.
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- One cycle of PCR yields 2 copies of target DNA sequence.
- A standard reaction of 30 cycles would yield 2^30 copies of DNA.
IMPORTANT!
- Know gene of interest
- Know its sequence
RESTRICTION ENZYMES
EXONUCLEASES
Cuts on the side of DNA molecules
ENDONUCLEASES
Cuts between DNA molecules
DNA Profiling
- Crime scene investigations or paternity tests.
- Banding patterns show up for each DNA sample and can be compared
- Fluorescent markers bind to codons in DNA fragments, so results can be seen.
- Horizontal gel electrophoresis in DNA profiling (charge and size)
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VARIABLE NUMBER TANDEM REPEATS (VNTRs)
- Short base sequences that show variation between individuals in terms of the number of repeats
- Example of highly repetitive sequences
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Genetic Engineering
- All living things use the same bases and the same genetic code.
- Each codon produces the same amino acid in transcription and translation, regardless of species.
- Sequence of amino acids in a polypeptide remains unchanged.
- We can take genes from one species and insert them into the genome of another species.
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GENE TRANSFER
(plasmid + host cell + restriction enzyme (endonucleases) + ligase)
1. Restriction enzyme cuts desired gene from genome
2. Same restriction enzyme cuts into plasmid (from bacterium)
3. Ligase joins sticky ends of desired gene and plasmid
4. Recombinant plasmid inserted into host cell; expresses new gene (e.g. human insulin production)
GMOs
Cloning
- A group of genetically-identical organisms derived from a single parent cell.
- Asexual reproduction always produces clones.
- In early stages of embryo development when cells are still pluripotent, an embryo can be fragmented or split to create animal clones.
SOMATIC CELL NUCLEAR TRANSFER (SCNT)
1. Somatic cells are taken from an adult organism to be cloned and grown in a low-nutrient medium. This inactivates genes to wipe out any previous patterns of differentiation.
2. Unfertilized egg cells are taken fro ma female of the same species and their nuclei are removed.
3. Cultured somatic cells and enucleated egg cells are placed side by side and zapped with a small electric pulse to fuse them together
4. Fused egg cells containing somatic cell nucleus develop into embryos for seven days, and then are implanted into surrogate mother.
