Polymerase Chain Reaction

Mass-Producing DNA

Starting with a very limited quantity of DNA, which may be contained in an impure sample, and using a test tube, some basic reagents, and a source of heat, the polymerase chain reaction (PCR) makes possible the production of millions of purified copies of DNA in only several hours. Prior to the introduction of this procedure, reproduction of DNA was difficult to perform, involved its cloning in bacterial cells, and consumed weeks to complete. PCR was developed in 1983 by Kary Mullis, an American biochemist working at Cetus, a California biotechnology company. In 1991, the PCR patent was sold for $300 million, and two years later Mullis was a co-recipient of the 1993 Nobel Prize. 

The Southern blot method, a common laboratory procedure, is used for the detection of a specific DNA sequence in a DNAcontaining sample. Applications include showing genetic relationships, such as to establish paternity, or DNA fingerprinting. The method was named after its inventor, the British biologist Edwin Southern (b. 1938).

The PCR procedure involves three primary steps, conducted at different temperatures: In the first step, the double-stranded DNA sample is subjected to a high temperature that causes it to split into two pieces of single-stranded DNA. Each strand serves as a template of the sequence of the DNA to be copied. After the addition of a primer, the polymerase enzyme (Taq) moves along the template, reading it and assembling a copy of the double-stranded DNA molecule. This is repeated thirty to forty times in an automated cycler, with the number of copies of the template increasing in an exponential manner with each cycle. 

The applications of PCR range from molecular biological research to such applied forensic applications as crime-scene fingerprint analysis. More specifically, PCR has been used to create transgenic animals as models of human disease, to diagnose genetic defects, to detect the AIDS virus in human cells, to establish paternal relationships, and in criminal investigations to link persons of interest to samples of blood and hair. Evolutionary biologists have been able to generate large quantities of DNA from trace amounts found in fossil remains and from a 40,000-year-old frozen woolly mammoth. PCR analysis, for example, has revealed that red pandas are more closely related to raccoons than to great pandas.