The polymerase chain reaction (PCR) permits the scientist to locate the molecular equivalent of a needle in a haystack. The PCR was developed at the Cetus Corporation in 1984 by Kary Mullis (Nobel laureate in Chemistry in 1993). It allows the DNA technologist to reproduce a single strand of DNA to a billion identical copies in a few hours. (A cancer cell, known for its high reproductive ability, would require a month to accomplish the same feat.) The DNA probe can then be used with a greater degree of accuracy and yield a more reliable result.

To multiply a DNA molecule with the PCR, four materials are required: the target DNA molecule (the one to be amplified); short strands of known "primer" DNA that will tag and identify the segment to be copied and provide a foundation for beginning the replication process; DNA polymerase, an enzyme that directs DNA replication in living cells (and for which the reaction is named); and a mixture of nucleotides, the nucleic acid building blocks from which new DNA will be formed.

Once the materials have been assembled, the PCR involves three major steps, all performed over and over again in a highly automated PCR machine. In the first step, the target DNA is heated, thereby breaking the bonds that hold the two strands together and unwinding the molecule. Then, in the second step, the temperature is reduced and the primer molecules bind to and flank the target DNA, thus bracketing and identifying the area to be copied. The primers are the "start" and "stop" signals in the copying.

An overview of the polymerase chain reaction (PCR) analysis
(a) DNA is obtained from a cell and placed in a test tube with appropriate other materials.
(b) The enzyme DNA polymerase duplicates the target DNA millions of times. (r With multiple copies of the DNA present, the DNA probe can easily locate its complementary binding site.

In the third step, DNA polymerase catalyzes the formation of single-stranded DNA molecules using the target DNA strands as templates. The synthesis begins at the spot marked by the primer DNA, and as the enzyme moves along each strand, it reads the sequence of chemical bases, using it as a template for assembling a complementary strand and a new nucleotide chain (the "chain reaction"). The DNA polymerase used is a special heat-tolerant enzyme called Taq polymerase. It is derived from the thermophilic bacterium Thermus aquaticus, first isolated from an oceanic thermal vent by Thomas Brock in the 1980s. The enzyme used must be heat-tolerant because a heat-fragile enzyme would have to be replaced after each heating step. At the conclusion of step three, four DNA strands exist where there were once two.

As the process continues, the PCR is repeated thirty to sixty times, each cycle beginning with a reheating of the mixture (step 1). A cycle takes about one to two minutes, and each new DNA segment serves as a template for many additional copies. Thus, the number of copies of DNA increases geometrically. Where there were originally two strands of DNA, there are now millions or billions. Instead of looking for a needle in a haystack, the DNA technologist has essentially made a huge stack of needles. Figure below summarizes the process.

Details of the polymerase chain reaction (PCR)
(a) Heat is used to separate the double-stranded (ds) DNA molecule. In cycle 1, the technologist adds a mixture of nucleotides, an enzyme called polymerase, and a segment of DNA primer.
(b) The polymerase extends the primer segment with the available nucleotides and produces two ds DNA molecules. (r The process is repeated, and at the end of cycle 2, four ds DNA molecules are present. Repeating the process in cycle 3 yields a total of eight ds DNA molecules. The strands increase in number geometrically in future cycles.

But the PCR is not without problems. Chief among these is contamination. If any contaminating DNA is present, it is amplified along with the target DNA. To eliminate this possibility, great care must be taken in sample preparation. Such preparation tends to be labor intensive and costly. Moreover, the PCR is not quantitative - it can determine that a particular DNA segment is present but it cannot determine how much of it was originally present. And, because the process is so new, reproducible results may be difficult to obtain and a period of time for standardization will be required. Despite these drawbacks, the PCR used in conjunction with DNA probes holds great promise for the future.