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Polymerase Chain Reaction (PCR) in vitro gene cloning
It is a method of copying fragments of DNA and needs:
DNA to be copied
DNA polymerase an enzyme which joins nucleotides together (Obtained from bacteria in hot
spring, therefore it is tolerant to heat and so does not denature at high temperatures)
Primers complementary short sequences of nucleotides to the end of the DNA fragments,
prevent the two separate strands of DNA rejoining
Nucleotides adenine, cytosine, guanine and thymine
Thermocycler precise temperature variation by a computer controlled machine
3 stages that copy both separated strands simultaneously, this can then be repeated doubling the
amount each cycle
1) The DNA fragments, primers and DNA polymerase are placed into a vessel within the
thermocycler with the temperature increased to 95°C. This causes separation of the DNA
2) It is then cooled to 55°C which means that the primers can join to their complementary bases on
the DNA fragment. The addition (annealing) of the primers is important as they are the
starting sequences which allow DNA polymerase to copy the DNA.
3) The temperature is increased to 72°C which is the optimum temperature for DNA polymerase to
add complementary nucleotides along the separated DNA strands. It begins at the primers and
adds nucleotides in sequences until the end of the chain, this is DNA synthesis.
It is important for aspects of science and medicine such as in forensic examinations of blood, hair or
semen samples and then this information can be used for cross-matching.
In vitro and in vivo gene cloning:
In vitro In vivo
It is very fast, which is valuable especially
It would take days or weeks to produce the
when only a small amount of DNA is available
same quantity of DNA as in vitro.
e.g. at the scene of a crime.
It does not require living cells meaning that It involves culturing the bacteria or vector
complex culturing techniques are not needed. which is often a complex and long procedure
It involved no risk of contamination as the
It requires a very pure sample as any
restriction endonuclease can match the
contaminant DNA would also be multiplied
`sticky ends' together
At any one time, 20% of the DNA cloned is
It is very accurate as the DNA copied has
copied inaccurately although modern
very few, if any, errors.
techniques have now improved.