In no particular order...
RFLP
STANDS FOR: Restriction Fragment Length Polymorphism (or patterns)
IS USED FOR: Determining DNA matches through gel electrophorsis
sort of like gel electrophorsis... except COMPLETE DIGESTION ONLY
takes advantage of DNA's negative charge; puts nylon membrane with positively charged electrode behind it. The DNA from the gel will then transfer OUT of the gel and INTO the nylon membrane.
Once in the nylon membrane, the membrane must be copied via x-ray in order for the DNA to be expressed.
"autoradiogram" is the final result.
Vector Cloning
- Used when wanting to express gene
- Antifreeze in fish expressed in tomato is a good example
1. isolate gene
2. cut with scissors (restricton enzymes)
3. attach gene of interest by sticky ends into plasmid
4. Inject plasmid into bacteria
5. "Infect" targets with new bacteria, thus expressing the gene in another living object.
PCR
PCR cloning is used in the laboratory; in vitro
- DNA is heated to remoe hydrogen bonds, forming 2 single strands
- The DNA primers are then attached, acting as RNA primers during DNA replication in nature
- The Taq polymerase then comes in and acts as the DNA polymerase III found in nature.
- The segments form 5-3; copy 3-5
- The DNA then starts replicating at an exponential rate
- Does not start targeting until after third copy.
After 29-30 cycles, it reaches 1 million copies; requires only one day
DNA Sequencing:
- Discovered by Sanger
- Similar to Gel electrophoresis
- Takes advantage of negative charge of DNA
- Requires Nylon Membrane to be x-rayed
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