Polymerase chain reaction
Polymerase chain reaction (Forensic Science)
The molecule of life, DNA (deoxyribonucleic acid), is the unique genetic blueprint of an individual. DNA is present in virtually every cell in the body. The uniqueness of DNA, which is composed of two long strands, stems from the specific order (sequence) of its different nucleotide building blocks, which are linked together to form each strand. Because the nucleotide sequence of each person’s DNA is unique (the only exception being identical twins, who have identical DNA), DNA analysis is an integral part of forensic investigations. DNA may be isolated from biological samples (such as blood, skin, semen, or hair) found at the scenes of mass disasters or crimes, from cheek cells swabbed from the insides of mouths (as may be taken from the parties in paternity determinations or from criminal suspects), or from the environment (as in the case of pathogens).
The specific sequence of nucleotides in several regions of the isolated DNA (short tandem repeats, known as STRs, or other specific sequences) is compared with the sequence in identical regions of a DNA standard. This standard comes from a known source; it may be from a predeath biological sample recovered from a disaster victim’s personal effects, from a crime suspect, from a party involved in a paternity case, or, in the case of a pathogen, from a previously experimentally derived sequence. When a crime is being investigated, the standard may be on file in the Federal Bureau of...
(The entire section is 543 words.)
Further Reading (Forensic Science)
Polymerase chain reaction
The Development of the Polymerase Chain Reaction (Genetics & Inherited Conditions)
The polymerase chain reaction (PCR) was developed by Kary B. Mullis in the mid-1980’s. The technique revolutionized molecular genetics and the study of genes. One of the difficulties in studying genes is that a specific gene can be one of approximately twenty-one thousand genes in a complex genome. To obtain the number of copies of a specific gene needed for accurate analysis required the time-consuming techniques of molecular cloning and detection of specific DNA sequences. The polymerase chain reaction changed the science of molecular genetics by allowing huge numbers of copies of a specific DNA sequence to be produced without the use of molecular cloning. The tremendous significance of this discovery was recognized by the awarding of the 1993 Nobel Prize in Chemistry to Mullis for the invention of the PCR method. (The 1993 prize was also awarded to Michael Smith, for work on oligonucleotide-based, site-directed mutagenesis and its development for protein studies.)
(The entire section is 152 words.)
How Polymerase Chain Reaction Works (Genetics & Inherited Conditions)
PCR begins with the creation of a single-stranded DNA template to be copied. This is done by heating double-stranded DNA to temperatures near boiling (about 94 to 99 degrees Celsius, or about 210 degrees Fahrenheit). This is followed by the annealing (binding of a complementary sequence) of pairs of oligonucleotides (short nucleic acid molecules about ten to twenty nucleotides long) called primers. Because DNA polymerase requires a double-stranded region to prime (initiate) DNA synthesis, the starting point for DNA synthesis is specified by the location at which the primer anneals to the template. The primers are chosen to flank the DNA to be amplified. This annealing is done at a lower temperature (about 30-65 degrees Celsius, or about 86-149 degrees Fahrenheit). The final step is the synthesis by DNA polymerase of a new strand of DNA complementary to the template starting from the primers. This step is carried out at temperatures about 65-75 degrees Celsius (149-167 degrees Fahrenheit). These three steps are repeated many times (for many cycles) to amplify the template DNA. The time for each of the three steps is typically one to two minutes. If, in each cycle, one copy is made of each of the strands of the template, the number of DNA molecules produced doubles each cycle. Because of this doubling, more than one million copies of the template DNA are made at the end of twenty cycles.
The PCR reaction is...
(The entire section is 293 words.)
Impact and Applications (Genetics & Inherited Conditions)
PCR is extremely rapid. One billion copies of a specific DNA can be made in a few hours. It is also extremely sensitive. It is possible to copy a single DNA molecule. Great care must be taken to avoid contamination, however, for even trace contaminants can readily be amplified by this method.
PCR is a useful tool for many different applications. It is used in basic research to obtain DNA for sequencing and other analyses. PCR is used in disease diagnosis, in prenatal diagnosis, and to match donor and recipient tissues for organ transplants. Because a specific sequence can be amplified greatly, much less clinical material is needed to make a diagnosis. The assay is also rapid, so results are available sooner. PCR is used to detect pathogens, such as the causative agents for Lyme disease or for acquired immunodeficiency syndrome (AIDS), that are difficult to culture. PCR can even be used to amplify DNA from ancient sources such as mummies, bones, and other museum specimens. PCR is an important tool in forensic investigations. Target DNA from trace amounts of biological material such as semen, blood, and hair roots can be amplified. There are probes for regions of human DNA that show hypervariability in the population and therefore make good markers to identify the source of the DNA. PCR can therefore be used to evaluate evidence at the scene of a crime, help identify missing people, and resolve paternity cases....
(The entire section is 240 words.)
Further Reading (Genetics & Inherited Conditions)
Budowle, Bruce, et al. DNA Typing Protocols: Molecular Biology and Forensic Analysis. Natick, Mass.: Eaton, 2000. Discussion includes DNA extraction and PCR-based analyses. Illustrations, bibliography, index.
Chen, Bing-Yuan, and Harry W. Janes, eds. PCR Cloning Protocols. Rev. 2d ed. Totowa, N.J.: Humana Press, 2002. Presents helpful introductory chapters with each section and guidelines for PCR cloning. Illustrations, bibliographies, index.
Dorak, M. Tevfik, ed. Real-Time PCR. New York: Taylor and Francis, 2006. Focuses on the practical aspects of PCR techniques, emphasizing how these methods can be used in the laboratory.
Guyer, Ruth L., and Daniel E. Koshland, Jr. “The Molecule of the Year.” Science 246, no. 4937 (December 22, 1989): 1543-1546. Reviews the “major scientific development of the year,” the polymerase chain reaction, noting that the technique, although introduced earlier, “truly burgeoned” in 1989.
Innis, Michael A., David H. Gelfand, and John J. Sninsky, eds. PCR Applications: Protocols for Functional Genomics. San Diego: Academic Press, 1999. Discusses gene discovery, genomics, and DNA array technology. Includes entries on nomenclature, expression, sequence analysis, structure and function, electrophysiology, and information retrieval. Illustrations, bibliography, index.
Kochanowski, Bernd, and Udo Reischl, eds....
(The entire section is 354 words.)
Web Sites of Interest (Genetics & Inherited Conditions)
Dolan DNA Learning Center, Biology Animation Library. http://www.dnalc.org/resources/animations/pcr.html. Viewers can watch an animated demonstration of PCR.
Library, University of California, Berkeley, PCR Project. http://sunsite3.berkeley.edu/PCR. An introductory overview to PCR prepared by professors and librarians at the university, including Paul Rainbow, an anthropology professor who wrote the book Making PCR: A Story of Biotechnology (1996). Provides access to several papers discussing the fundamentals of PCR, applications of PCR basics, and technical variations in basic PCR methods.
University of Utah, Genetic Science Learning Center, PCR Virtual Lab. http://learn.genetics.utah.edu/content/labs/pcr. The virtual lab provides a demonstration of PCR.
(The entire section is 111 words.)