Polymerase Chain Reaction

Brief Description:

PCR, or polymerase chain reaction, uses a thermostable DNA polymerase to amplify genomic sequences of living organisms via in vitro methods. Observations of PCR results can lead to information regarding genetic variances, chromosomal rearrangements, and the presence of viral pathogens (Saiki et al. 1988). PCR can also have applications in diagnostics and forensics (Leboffe & Pierce 2005).

The cell must be melted (heated to 95ºC) in order to separate the strands of the DNA template. Then the reaction is cooled to 55ºC and DNA primers can anneal (Slonczewski & Foster). Two oligonucleotide primers are used that are identical to the ends of the targeted DNA sequence by the DNA polymerase to attach nucleotides that are complementary to the target (Saiki et al. 1988). The polymerase used is called Taq, from the thermophile Thermus aquaticus, which uses primers to replicate the target DNA sequence. The primers act as limits so that Taq does not amplify more than the desired sequence, which will then be used for identification. Taq is useful in this reaction because it is not denatured at the high temperatures of PCR (Leboffe & Pierce 2005).

Extension occurs at 72ºC when Taq extends the primers by adding nucleotides to the template (Leboffe & Pierce 2005). The process of heating and reannealing the single strands involves the creation of multiple target strand replications (Slonczewski & Foster)Samples are then loaded into some type of polyacrylamide gel, where electrophoresis runs and results can be viewed with illumination by UV light (Lavallée et al. 1994).

PCR can be used in many circumstances. It has applications in cloning, and it can be used to detect the presence or absence of an organism in a particular environment such as wine, described below (Slonczewski & Foster).

Thermocyclers are the equipments used in PCR. They can be programmed for the appropriate temperatures and times to eliminate the tedious use of water baths and manually transferring the samples (Leboffe & Pierce, 2005).

RAPD-PCR is a method using short primers that hybridize at a variety of locations on the genome. It is a variant of PCR aimed at the amplification of multiple fragments of DNA (Quesada & Cenis 1995).

Application in Wine Microbiology:

In the identification of wine yeast, the 600nts D1/D2 loop is a region with highly conserved ends in yeast that may be used to discriminate most genera. Thus PCR can be used to identify the wild yeast present in a wine, used to determine the cause for a spoiled wine. In the study by Quesada and Cenis (1995), RAPD-PCR is used to amplify yeast DNA for identification of a spoilage species. This is particularly useful because it saves time and conclusions can be made regarding winemaking microbiology as soon as possible. Despite errors of reproducibility on some occasions, RAPD-PCR provides the ability to partially sequence genomes for identification without previous knowledge of the microbe we are looking for (Quesada & Cenis 1995).

Since the range of wine yeast strains has become so large, accurate identification requires very specific technique in order to enforce quality control. Not only can the identity of commercial yeasts be identified with PCR, but the reaction can also be used to determine the stability of the yeast strains during propagation and storage (Lavallée et al. 1994).

References:

  • Lavallée, F., Salvas, Y., Lamy, S., Thomas, D.Y., Degré, R., Dulau, L. 1994. PCR and DNA fingerprinting used as quality control in the production of wine yeast strains. Am. J. Enol. Vitic. 45(1): 86-90.
  • Leboffe, M.J. and Pierce, B.E. 2005. A photographic atlas for the microbiology laboratory. Morton Publishing Company; 83-85.
  • Quesada, M.P., & Cenis, J.L. 1995. Random amplified polymorphic DNA (RAPD-PCR) in the characterization of wine yeasts. Am. J. Enol. Vitic. 46(2): 204-207.
  • Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B., and Erlich, H.A. 1988. Primer-directed amplification of DNA with a thermostable DNA polymerase. 239: 487-491.
  • Slonczewski, J.L. & Foster, J, W. 2009. Microbiology: an evolving science. W.W. Norton & Co.: New York. 250.

Tags