Biotechnology 2.0 Flashcards
U2L6b
What is the Polymerase Chain Reaction?
The PCR reaction is a method scientists use to isolate and amplify a gene of interest.
What Polymerase does PCR use?
PCR uses Taq polymerase which is isolated from Thermus Aquaticus.
The polymerase works best at 72C and wont denature at 95C
How is the PCR reaction setup?
The identified gene of interest is located in a genome. RNA primers then get produced to bind to the sample DNA on either side of the gene.
The sample is then thrown into a test tube with Taq Polymerase, the RNA primers and A,C,T,G. The tube gets heated and cooled in a PCR machine.
How does the PCR heat cycle work?
@ 95C - DNA strand is separated into two
@60C - Primers bind to the DNA
@72C - Taq Polymerase gets added and begins duplicating the DNA with the target gene.
This cycle repeats 30 times in an hour.
How many copies of a gene does PCR make after an hour?
PCR has the capacity to produce more than 60 billion copies of the target gene after just one hour of Heat cycling the original DNA.
What are Plasmids?
Plasmids are small circular sections of DNA that are found in Bacterial Cells. Genes in Plasmids do not directly affect the function of a Bacteria but the bacterial cell can express the genes that are found in the plasmid.
Plasmid will also get replicated when cell replicates.
How is foreign DNA inserted into the Plasmid?
Plasmid and target gene are cut out with restriction enzymes.
The cut target gene and plasmid are then mixed together in a test tube with ligase to merge everything together.
What happens after the target gene is inserted into the Plasmid?
The Plasmids are placed into a test tube with Bacteria. The bacteria are treated with a chemical that makes the cell wall more porous and more likely to accept the modified plasmid.
What are the three possible outcomes?
1) Bacteria will die due to antibiotic exposure
2) Bacteria will survive but no trace of Plasmid will be present
3) Bacteria will survive with Plasmid.
What is the name of a bacteria that successfully expressed the target gene?
It is called a “Recombinant Bacteria”.
