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In vitro gene cloning (the polymerase chain reaction)
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Notes covering in vitro cloning, the PCR, Advantages of in vitro and in vivo cloning
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Abi StarrChapter 16.3 In vitro gene cloning – the polymerase chain reaction
Polymerase chain reaction
The PCR is a method of copying fragments of DNA. The process is automated, making it fast and
efficient. The process requires the following:
The DNA fragment that is to be copied
DNA polymerase – the enzyme that can join nucleotides together. It is obtained from
bacteria in hot springs and is therefore tolerant to heat (thermostable) and does not
denature during the high temperatures used in the process
Primers – short sequences of nucleotides that have a set of bases complementary to those
at one end of each of the two DNA fragments
Nucleotides – which contain each of the four bases found in DNA
Thermocycler – A computer controlled machine that varies temperatures precisely over a
period of time
The PCR is carried out in three stages:
1. Separation of the DNA strand - The DNA fragments, primers and DNA polymerase are
placed in a vessel in the thermocyler. The temperature is increased to 95°C, causing the two
strands of the DNA fragment to separate
2. Addition (annealing) of the primers – The mixture is cooled to 55°C, causing the primers to
join (anneal) to their complementary bases at the end of the DNA fragment. The primers
provide the starting sequences for DNA polymerase to begin copying DNA because DNA
polymerase can only attach nucleotides to the end of an existing chain. Primers also prevent
the two separate strands of DNA from rejoining
3. Synthesis of DNA – The temperature is increased to 72°C. This is the optimum temperature
for DNA polymerase to add complementary nucleotides along each of the separated DNA
strands. It begins at the primer on both strands and adds the nucleotides in sequence until it
reaches the end of the chain.
Because both separated strands are copied simultaneously there are now two copies of the original
fragment. Once the two DNA strands are completed, the process is repeated by exposing them to
the temperature changes again, which results in four strands. The whole cycle takes around 2
minutes. Over a million copies of the DNA fragment can be made in just 25 cycles and 100billion
copies can be produced in just a few hours. Even the smallest sample of DNA from a single hair r
drop of blood can be multiplied to allow forensic examination and accurate cross matching.
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