Unlike gene cloning, Polymerase Chain Reaction is carried out in a test tube. DNA is mixed with reagents and the test tube is placed in a thermal cycler. Thermal cycler enables the mixture to be incubated at a series of temperatures that are varied in a preprogrammed manner.

Following are the steps:

1. Heat the mixture to 94 degree Celsius to break H bonds that hold the double strand together. This is called denaturation.
2. Cool the mixture to 50-60 degrees. The strands can join back together at this temperature but they won't due the heavy presence of oligonucleotides or primers which anneal to the DNA molecules at specific positions.
3. Temperature is raised to 74 degrees, which is optimum temperature for Taq DNA polymerase which is present in the mixture. Taq DNA polymerase attaches to one end of each primer and synthesizes new strands of DNA, complementary to the template DNA molecule. Thus we now have 4 strands instead of 2.
4. We then increase the temperature back to 94 degrees and denature to repeat the process. At the end of this step, we would have 8 DNA strands. We continue until, the DNA is sufficiently amplified.

Gene Isolation

Gene isolation is often a prerequisite to studying the gene. Gene cloning allows us to isolate genes. This is done by fragmenting the DNA into pieces and checking if a gene or gene fragment is present in the section.

PCR can also be used to isolate genes. If the primers are placed at the boundaries of a gene, the gene would be duplicated much more efficiently than by gene cloning. However, selecting the right boundaries is often difficult. Thus PCR cannot be used to study genes which have not been studied before. In addition, PCR cannot be used on gene longer than 40 kb.

Gene cloning is the only method which can be used with unknown genes or long genes.

Source

Gene Cloning & DNA Analysis by T. A. Brown