Polymerase Chain Reaction (PCR)
Polymerase chain reaction (PCR) has been used as an alternative to cloning.
This technique can be used for amplification of DNA segments of which the end
sequences are known. It is a simple and fast method of amplifying a DNA segment
into a large pool of DNA.
In this method restriction enzymes are used to digest a segment of DNA.
Then heat is used to denature the double strand. Since the sequence of
the end parts of the strands are known, RNA primers can be attached to
the 3' ends of the DNA segment and the temperature is lowered to 50-60°.
Using a DNA polymerase enzyme and an added pool of deoxynucleotides, the DNA
segment between the two primers is reproduced. When this is completed the mixture
is heated again to 90o to denature the strands and continue with a new cycle. Each
time this cycle is repeated, the number of new strands produces doubles and causes
an exponential increase in the number of strands.
This process is highly effective and has been used for analyses of mutations,
DNA damage or other sequence related phenomena of the genome.