The polymerase chain reaction (PCR) was introduced in 1985 by Saiki et al. (1), who recognized that sequence-specific oligonucleotides could be used to prime synthesis of complementary DNA strands by any of several DNA polymerases and that these primers could be chosen so as to replicate both strands of the DNA positioned between their cognate sequences in the template (1,2). Initial studies with the technique were laborious and thus limited in scope. Thermostable DNA polymerases were not yet commercially available, so the experimenter had to add fresh polymerase after the DNA denaturing step of each cycle (1,2). The process was not automated, so a student or technician was needed to transfer the reactions between water baths or heating blocks set for each temperature of the cycle. The original report was largely a “proof of principle” study in which a portion of the human ß-globin gene was amplified and then analyzed for the presence of normal versus sickle cell coding sequences (1,3). In 1988, the use of a thermostable DNA polymerase, isolated from Thermus aquaticus, was used in this procedure instead of the Escherichia coli Klenow fragment (4,5). This modification constituted the major advance needed to make the technique practical and commercially viable. Various preparations of heat-stable polymerases from T. aquaticus, as well as other thermophilic bacteria, were soon commercially available, as were several models of programmable instruments for rapid and reliable heating and cooling of samples.
KeywordsPolymerase Chain Reaction Product Thyroid Papillary Carcinoma Microcentrifuge Tube Reverse Transcription Reaction Upstream Primer
Unable to display preview. Download preview PDF.
- 23.Lundberg, K. S., Shoemaker, D. D., Adams, M. W., Short, J. M., Sorge, J. A., Marthur, E. J. (1991) High-fidelity amplification using a thermostable DNA polymerase isolated from Pyrococcus furiosus. Gene 108, 1–6.Google Scholar