10 Things You Need to Know About BIOTECHNOLOGY!

10. Vector Cloning (5 steps)
1. Isolate the desired genes in the plasma and human DNA
2. Insert human DNA into plasmid
3. Plasmid is placed into bacteria through transformation
4. Bacteria replicates
5. Identify the clone that carries the DNA

9. Sticky Ends & Blunt Ends
- Sticky ends are in a staggered formation, and will allow the formation of hydrogen bonds with other DNA molecules that were cut with the same enzyme
- Blunt ends will not form this hydrogen bond

DO NOT USE ENZYMES THAT CREATE BLUNT ENDS (the ends will not be able to stick together)
- Ligase is used to create phosphodiester bonds that secure the DNA fusion


8. PCR (Polymerase Chain Reaction)
- Used when DNA is contaminated or very limited
- Extremely fast
- Does not require the use of any other cells
- Three Step Cycle: Heating, Cooling, and Replication
- THe DNA is incubated in a test tube with a solution of Taq Polymerase, Nucleotides, and Primers
- Heating the DNA segment induces Denaturation, which separates them into single strands of nucleotides
- Taq Polymerase elongates the strand creating its complementary strand
- Cooling the test tube binds the single stranded DNA from heating into a double-stranded  DNA

7. RFLP (Restriction Fragment Length Polymorphism)
- Uses restriction enzymes to separate DNA molecules into shorter fragments
- The fragments are differentiated and organized through Gel Electorphoresis
- DNA sequences may differ in one or more restriction sites, thus they may produce fragments of different sizes

6. Autoradiography
- Nucleic acid hybridization will use a specific probe to identify and label bands that are related to the gene of interest
- A radioactive label is placed, identifying the target fragments
- Southern Blotting transfers the patterns on the gel to a sheet of nitrocellulose paper
- Soaking the sheet into the solution with the probe allows us to visualize the bands


5. Gel Electrophoresis
- Separates macromolecules (nucleic acids + proteins) based on rate of movement, which is determined by their size, electrical charge and other properties
- The Gel Electrophoresis moves from negative to positive
- Smaller fragments are able to travel further along the Gel, while larger fragments tend to stay behind

4. DNA Sequencing
- Uses dideoxynucleotides
- ddATP, ddCTP, ddTTP, ddGTP
- Stops elongation when mixed with a growing DNA strand
- Fragments of various lengths are made
- Gel Electrophoresis is used to determine the order of the nucleotide sequence

3. Restriction Enzymes
- Cut DNA molecules at a specific point in the sequence
- EcoRI
- SmaI
- AluI
- SalI  (blunt end)
- HindIII (blunt end)
- PstI

2. Incomplete & Complete Digestion
- Enzymes that undergo incomplete digestion are less expensive
- Complete digestion will make the restriction enzymes cut at all cut sites
- Incomplete digestion will make restriction enzymes cut at random cut sites (or none at all)

1. Transcription / Translation
- Know how to transcribe and translate
- Review exons / introns
- Know how to isolate the desired gene