The Polymerase Chain Reaction or PCR
The polymerase chain reaction (PCR) has made a significant impact in modern molecular biology and molecular medicine. Therefore, we now live in the time after PCR since many diagnostic tests now use PCR. The image below illustrates sample sources of nucleic acid templates for PCR as well as a sample of derived analytical methods.
PCR analysis can be performed using DNA or cDNA (RNA). A variety of sources are available for the preparation of the nucleic acid templates, including forensic samples. PCR now forms the basis for numerous analytical techniques. Most methods utilize differential amplification of target sequences, but other methods may involve advanced applications for the analysis of PCR products.
The Polymerase Chain Reaction (PCR) is a technique that allows making many copies of a piece of DNA in a laboratory using readily available reagents. During the reaction, the number of copies increases exponentially. Therefore, within a few hours more than 100 billion copies of a DNA piece can be made. The availability of DNA polymerases and synthetic oligo-nucleotides made this technique possible. PCR has now become an alternative to cloning since it allows amplifying specific sequences in a complex mixture. In addition, PCR combined with sequencing techniques allows identification of mutant alleles in a DNA sample specifically, rapidly and with high sensitivity.
The PCR technology allows specifically amplifying a target DNA sequence from a tiny amount of starting material. In earlier versions of PCR the Klenow fragment of E. coli DNA polymerase, I was used. However, the Klenow fragment is not thermo-stable. The introduction of a thermo-stable DNA polymerase, such as the DNA polymerase found in Thermus aquaticus, resulted in a major technological breakthrough in the development of PCR methodologies.
PCR can be categorized into different groups or methods based on how the techniques are used. However, all these methods follow the same basic steps and principal.
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Figure 1: Models of TAQ polymerase. Three different depictions of the same model of TAQ polymerase are illustrated (Eom et al. 1996).
Eoam, Wang, and Steitz solved the co-crystal structure of Taq polymerase with a blunt-ended duplex DNA bound to the polymerase active-site cleft. The structure indicates that the DNA neither bends nor goes through the large polymerase cleft. The structural conformation of the bound DNA is between the B and A forms. The model of the structure showed that a wide minor groove allows access to protein side chains. The side chains are hydrogen-bonded to the N3 of purines and the O2 of pyrimidines at the blunt-end terminus.
DNA polymerases catalyze the synthesis of long polynucleotide chains. The synthesis starts from monomeric deoxynucleotide triphosphates in the presence of an original parental DNA strand. The DNA strand serves as a template for the synthesis of a new complementary DNA strand. The synthesis proceeds in the 5’ to 3’ direction. The polymerization occurs from the 5’ α-phosphate of the deoxynucleoside triphosphate to the 3’ terminal hydroxyl group of the growing DNA strand. DNA polymerases require a short segment of DNA to anneal to a complementary sequence. This DNA sequence or oligonucleotide is called a primer since it is needed to prime the synthesis.
The Polymerase Chain Reaction or PCR was discovered, conceived or invented by Kary B. Mullis in 1983. According to him he stumbled upon this reaction when driving in northern California during a moonlit night. Furthermore, as pointed out by him, the reaction allows generating up to 100 billion similar DNA molecules from a single DNA molecule within a few hours. The reaction can be performed in a test tube but also requires a few reagents and a source of heat. The introduction of the PCR reaction has made life much easier for molecular biologists, allowing them to produce as much DNA as they want. The technology has spread throughout the biological sciences with tremendous speed. However, since the reaction involves thermal cycling the most important piece of the ultimately improved PCR technology turned out to be the use of a heat stable DNA polymerase. The polymerase that was originally extracted from the bacterium Thermus aquaticus is now used in almost all PCR reactions. Thermus aquaticus lives in hot springs. The polymerase chain reaction has become the ultimate game-changing technology in molecular biology. In 1993 Kary B. Mullis was awarded the Nobel Prize in Chemistry “for contributions to the developments of methods within DNA-based chemistry” jointly with Michael Smith. Kary B. Mullis received one-half of the prize for his invention of the PCR method. Michael Smith received the other half for his contributions to oligonucleotide-based, site-directed mutagenesis and development for protein studies. {http://www.nobelprize.org/nobel_prizes/chemistry/laureates/1993/}.
Dr. Mullis now has his own website: http://www.karymullis.com/
Features of the polymerase chain reaction
- The Polymerase Chain Reaction (PCR) selectively amplifies a target DNA molecule.
- It allows the extension of short single-stranded synthetic oligonucleotides, primers, during repeated cycles of heat denaturation, primer annealing, and primer extension.
- PCR is a cyclic process in which a sequence of steps is repeated over and over again.
- Double-stranded fragments of DNA are separated into single strands by mild heating called denaturation.
- A short, synthetic oligonucleotides primer is added to the reaction mixture, along with the four deoxyribonucleotide triphosphates dATP, dGTP, dCTP, dTTP, and a DNA polymerase now usually a Taq DNA polymerase.
- DNA polymerase catalyzes the synthesis of complementary new strands.
Reference
Eom SH, Wang J, Steitz TA.; Structure of Taq polymerase with DNA at the polymerase active site. Nature. 1996 Jul 18; 382(6588):278-81.
Faloona, F., Weiss, S., Ferre, F., and Mullis, K. 1990. Direct detection of HIV sequences in blood high-gain polymerase chain reaction [abstract]. In: 6th International Conference on AIDS, University of California, San Francisco: San Francisco (CA). Abstract 1019.
Gelfand, D.H. and White, T.J. 1990. Thermostable DNA polymerases. In: PCR Protocols: A Guide to Methods and Applications. Innis, M.A., Gelfand, D.H., Sninsky, J.J., and White, T.J., eds. San Diego: Academic Press. 129–141.
Holland, P.M., Abramson, R.D., Watson, R., and Gelfand, D.H. 1991. Detection of specific polymerase chain reaction product by utilizing the 5´→3´ exonuclease activity of Thermus aquaticus DNA polymerase. Proc. Natl. Acad. Sci. USA 88:7276–7280.
Innis, M.A., Myambo, K.B., Gelfand, D.H., and Brow, M.A. 1988. DNA sequencing with Thermusaquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA. Proc. Natl. Acad. Sci. USA 85:9436–9440.
Mullis K. In Methods In Enzymology, Vol.155, 335, 1987.
Kary B. Mullis; The Unusual Origin of the Polymerase Chain Reaction. SCIENTIFIC AMERICAN April 1990. 56-65.