21.3 in vitro gene cloning - PCR reaction Flashcards
what are the 5 components needed for PCR?
1) DNA fragment
2) taq polymerase (DNA polymerase)
3) primers
4) nucleotides (all bases)
5) thermocycler
why are taq polymerase used and where are they found?
found in hot springs; thermostable & does not denature
what are primers?
short dna sequences with bases complementary to the end of dna fragments
why are nucleotides needed for PCR reactions?
dna synthesis
what is a thermocycler?
computer controlled machine that varies temperature
give 2 advantages of in vitro cloning.
1) extremely rapid - useful for crime scene forensics
2) no living cells needed
what is a disadvantage of using in vitro cloning?
contaminating DNA is amplified as well -> gives false results
what are the 3 steps of PCR?
1) denaturation
2) annealing
3) elongation
what temperature is needed in denaturation and what occurs?
95°C
- 2 strands of dna separates due to breaking of hydrogen bonding
what is the temperature needed for annealing and what occurs?
53°C
mixture is cooled & primers anneal to complementary bases at the end of dna fragments
- primers provide starting sequence for taq polymerase & prevent 2 strands from re-joining
what temperature is needed for elongation & what happens during it?
72°C
nucleotides are added along dna strands
what is the optimum temperature for taq polymerase? which PCR stage does it correspond to?
72°C - elongation
