Polymerase, << PAHL ih muh rays, >> chain reaction, often called PCR, is an important scientific technique that enables scientists to rapidly and precisely copy genes and genetic material. The technique can generate large amounts of any piece of DNA from any organism. DNA—deoxyribonucleic acid—is a double-stranded chainlike molecule found in every living cell. It directs the formation, growth, and reproduction of cells and organisms.
The DNA molecule.
All DNA consists of thousands of smaller chemical units called nucleotides. Nucleotides are chemically bonded to one another to form a double-stranded chain of DNA. Each nucleotide contains a compound called a phosphate, a sugar called deoxyribose, and a compound called a base. The phosphate and sugar are the same in all DNA nucleotides, but the bases vary. There are four DNA bases: (1) adenine, (2) guanine, (3) thymine, and (4) cytosine.
DNA bases are paired in a specific way. For example, an adenine base on one strand of a double-stranded segment of DNA always bonds with a thymine base on the opposing strand. Similarly, guanine on one strand always bonds with cytosine on the other. Because the sequence of bases in one strand is determined by the sequence of bases in the other, scientists say the two chains are complementary.
The PCR process.
The main components of the PCR process are: (1) the double-stranded segment of DNA to be copied, (2) short, synthetic, single-stranded segments of DNA called primers, (3) a supply of the four nucleotides that make up DNA, and (4) a special kind of enzyme (a protein that speeds up a chemical reaction among other materials) called DNA polymerase. These components can all be combined before beginning the PCR process or they can be added at certain steps in the process. In addition, the segment of DNA to be copied can be part of a much larger piece of DNA. The PCR process will copy only the relevant portion.
The first step in the PCR process involves heating the DNA to about 200 °F (93 °C). The DNA segment to be copied can be thought of as consisting of two partnered (complementary) strands, Strand A and Strand B. Heating the segment causes the strands to separate, or “unzip.” Each separate strand becomes a template (pattern) for a new DNA chain to be created.
In the next step, the temperature is lowered to about 140 °F (60 °C) so that the primers can bind to each strand. The primers are artificial copies of small end portions of the separate strands. For example, the primer that binds to Strand A would be a manufactured copy of a short stretch of the complimentary end of Strand B. The primer binds with the complimentary end portion of Strand A, forming a partially duplicated version of the original double-stranded segment of DNA. However, most of Strand A remains single-stranded and un-partnered at the end of this step.
The temperature is then raised again to about 160 °F (71 °C). Around this temperature, DNA polymerase enzyme causes the added nucleotides to bind to the un-partnered portions of each strand. The polymerase interacts with unattached nucleotides and binds complimentary nucleotides to the remainder of the template. This process continues, creating a full copy of Strand B that is “zipped up” with Strand A. When the process is finished, the two single strands of the original DNA sample have been replaced by two identical double strands. Both the original sample and the newly synthesized double-stranded product can now act as templates for additional rounds of PCR.
The original chain of DNA to be copied may be a complicated mixture of thousands of nucleotides, but the primers and polymerases precisely duplicate the sequence. The steps in the PCR process, called a cycle, take only minutes to complete. Each cycle doubles the amount of DNA. The cycles can be repeated 30 or more times within a few hours. After 30 cycles, more than one billion copies of a single piece of DNA are produced. Using PCR, scientists can obtain large amounts of genetic material to analyze from even minute samples of blood, tissue, or other substances from organisms.
History.
Kary B. Mullis, an American biochemist, first described the PCR technique in 1985. He had developed PCR while working at the Cetus Corporation, a biotechnology firm in California. Mullis won the Nobel Prize for chemistry in 1993 for inventing this revolutionary technique. In 1987, Cetus patented the process. PCR has since become one of the most important scientific technologies.
PCR is widely used in DNA fingerprinting to help identify crime suspects. It is also used to detect infectious disease organisms, such as bacteria and viruses, from small samples of tissue or fluids. Using PCR, medical researchers can identify harmful mutations in genes that lead to disease in human beings, such as cystic fibrosis. PCR is also useful for studying variations in DNA among human populations. Some biologists use PCR to compare genetic similarities between different species to understand evolutionary relationships among organisms.
See also DNA ; DNA fingerprinting ; Mullis, Kary Banks .
