[21.3] in vitro gene cloning - the polymerase chain reaction Flashcards
1
Q
what is PCR used for?
A
to automatically copy fragments of DNA
2
Q
what is needed in the PCR process? (5)
A
- DNA fragment to be copied
- DNA polymerase
- primers
- nucleotides
- thermocycler
3
Q
what specific DNA polyermase is needed for PCR?
A
- thermostable DNA polymerase which is able to tolerate high temperatures without denaturing
- obtained from bacteria in hot sulfur springs which have a greater proportion of cysteine AAs so more disulfide bridges, strengthening its tertiary structure
4
Q
what are primers?
A
short sequences of nucleotides that have a set of bases complementary to those at one end of the two DNA fragments
5
Q
why are primers needed in PCR?
A
- prevents the two complementary strands from rejoining
- provides starting sequences for DNA polyermase to begin copying DNA as DNA polyermase can only attach nucleotides to the end of an existing chain
6
Q
what is a thermocycler?
A
computer-controlled machine that varies temperature precisely over a period of time
7
Q
what are the 3 stages of PCR?
A
- separation of DNA strands
- addition of primers
- synthesis of DNA
8
Q
describe what happens in the 1st stage of PCR
A
- DNA fragments, primers, and DNA polymerase are placed in vessel in thermocycler
- double-stranded DNA is heated to 95°C
- H bonds between the two strands are broken, separating the strand of DNA fragment
9
Q
describe what happens in the 2nd stage of PCR
A
- mixture is cooled to 55°C
- primers to join to their complementary bases at the end of the DNA fragment
10
Q
describe what happens in the 3rd stage of PCR
A
- temperature is increased to 72°C
- optimum temperature for DNA polyermase to add complementary nucleotdies along each of the separated DNA strands
- begins at primer on both strands and adds nucleotides in sequence until it reaches the end of the chain
11
Q
what are the advantages of in vitro gene cloning?
A
- extrememly rapid, which is particularly valuable if there is only a small amount of DNA available
- doesn’t require living cells
