User:Ruud Koot/Nupedia/Polymerase Chain Reaction (brief version)

PCR

Polymerase Chain Reaction (PCR) is a method for amplifying (creating multiple copies of) DNA without using a living organism such as E. coli or yeast. Kary Mullis invented the PCR process in the early 1980s and was later awarded the Nobel Prize in Chemistry for his work. In 1989, Hoffman La Roche and Perkin-Elmer Corporation patented this process. PCR is commonly used in medical and biological research labs for a variety of tasks, such as the detection of hereditary diseases, the identification of genetic fingerprints, the building of DNA-based phylogenetic trees (trees of species relations), the cloning of genes, and paternity testing.

PCR can start with a very small amount of template DNA (original DNA), at times, with just one strand. During the PCR process, the given DNA is copied by an enzyme called DNA-Polymerase, which duplicates a DNA strand. Usually, only a small part of the DNA strand is copied using PCR. This part is selected by primers, short artificial DNA strands (20-40 base pairs),^N which exactly match each end of the part to be copied.

The PCR machine is basically a computer-controlled oven, where a program controls the timing and the temperature. The PCR process consists of several iterations, usually 15-30, and each iteration consists of the following steps (Figure 1):

1. Melting (96°C, 30-600 seconds)
   In the melting step, the double-stranded DNA is divided into two single strands, like opening a zipper. In the current practice of PCR (April 2001), the DNA-Polymerase is thermostable (stable at high temperatures). Since the DNA-Polymerase is currently derived from bacteria in geysers, it is not inactivated by the melting step.
2. Annealing (65-80°C, 30-120 seconds)
   In the annealing step, the primers "anneal" (attach themselves) to the single DNA strands. The DNA-Polymerase then binds to each annealed primer.
3. Elongation (65-80°C, 30-120 seconds)
   In the elongation step, the DNA-Polymerase runs along the single DNA strand, creating the missing second DNA strand in the process.

After each iteration, the amount of DNA is doubled. Therefore, after multiple iterations, the amount of DNA has increased by powers of 2. For example, after 30 iterations one DNA strand has been copied into 2³⁰ = 1 073 741 824 strands that are exact copies of the part of the first strand that was chosen by the primers.

Figure 1 : Schematic drawing of the PCR cycle.^N
(1) Melting at 96°C. (2) Annealing at 68°C. (3) Elongation at 72°C (P=Polymerase). (4) The first cycle is complete. The two resulting DNA strands make up the template DNA for the next cycle, thus doubling the amount of DNA duplicated for each new cycle.

With PCR it has become possible to "clone a gene," just one gene or even just a part of a gene. This process should not be confused with the cloning of a whole organism. "Cloning a gene" involves, first, the separating of a single gene from one organism, and then, the inserting of the gene into another organism, like a bacterium. As a result, the cloned gene can then be studied in detail in the new organism. PCR is often used in this process to isolate a single gene through amplification, before it is transferred to the new organism. PCR is also used to introduce mutations (changes) into strands of DNA. Through PCR mutation, then, a gene can be mutated to study the effects of such a change. PCR is an important tool in biotechnology. For example, it is used to alter bacteria for the purpose of producing medicine.

For Further Reading

Saiki, R. K., D. H. Gelfand, S. Stoffel, S. J. Scharf, R. Higuchi, G. T. Horn, K. B. Mullis, and H. A. Erlich. "Primer-Directed Enzymatic Amplification of DNA with a Thermostable DNA Polymerase." Science 239 (1988): 487-491.
United States Patent 5,656,493 Mullis, et al. August 12, 1997: System for automated performance of the polymerase chain reaction