The Polymerase Chain Reaction (PCR): Cloning DNA in the Test Tube

The polymerase chain reaction is a technique for quickly "cloning" a particular piece of DNA in the test tube (rather than in living cells like E. coli). Thanks to this procedure, one can make virtually unlimited copies of a single DNA molecule even though it is initially present in a mixture containing many different DNA molecules.

The procedure

• In order to perform PCR, you must know at least a portion of the sequence of the DNA molecule that you wish to replicate.
• You must then synthesize primers: short oligonucleotides (containing about two dozen nucleotides) that are precisely complementary to the sequence at the 3' end of each strand of the DNA you wish to amplify.
• The DNA sample is heated to separate its strands and mixed with the primers.
• If the primers find their complementary sequences in the DNA, they bind to them.
• Synthesis begins (as always 5' -> 3') using the original strand as the template.
• The reaction mixture must contain
  • all four deoxynucleotide triphosphates (dATP, dCTP, dGTP, dTTP)
  • a DNA polymerase. It helps to use a DNA polymerase that is not denatured by the high temperature needed to separate the DNA strands.

  Discussion of DNA synthesis

• Polymerization continues until each newly-synthesized strand has proceeded far enough to contain the site recognized by the other primer.
• Now you have two DNA molecules identical to the original molecule.
• You take these two molecules, heat them to separate their strands, and repeat the process.
• Each cycle doubles the number of DNA molecules.

Using automated equipment, each cycle of replication can be completed in less than 5 minutes. After 30 cycles, what began as a single molecule of DNA has been amplified into more than a billion copies (2³⁰ = 1.02 x 10⁹).

With PCR, it is routinely possible to amplify enough DNA from a single hair follicle for DNA typing. Some workers have successfully amplified DNA from a single sperm cell. The PCR technique has even made it possible to analyze DNA from microscope slides of tissue preserved years before. However, the great sensitivity of PCR makes contamination be extraneous DNA a constant problem.

Supplementary reading

Mullis, K. B., "The Unusual Origin of the Polymerase Chain Reaction", Scientific American, April 1990. The author tells how he worked out the principles of the PCR (which won him a Nobel Prize) while driving at night to his weekend retreat in the California mountains.