Quantitative/Real-Time PCR

Brief Description:

Quantitative PCR was a major advancement in DNA identification that made it possible for very rapid identification and made many tedious practices previously used a thing of the past. Essentially, quantitative PCR exponentially amplifies a small DNA fragment (100-600 base pairs) within a longer strand of normal, double stranded DNA. Two primers are chosen that are complementary to a sequence of interest on each of the two strands of the original double stranded DNA. DNA polymerase is then used to copy from the primer down the strand to copy the defined sequence. These primers can then be used over and over again to copy both the original template but also the copies, enabling very rapid amplification. To make this process even more successful, a thermally stable DNA polymerase enzyme was isolated from Thermus aquaticus, a bacterium that grows in very hot environments. Since high temperatures are required in order to separate double stranded DNA, the use of Taq polymerase has negated the need to add new polymerase in every amplification process (Hunt).

By the end of the amplification process there will be a pool of the DNA fragment if the DNA of interest actually contains the sequence that is being amplified. However, the amount of DNA left over after amplification does not correlate precisely with the amount of DNA put into the reaction initially. For this reason, real-time PCR is better used as a qualitative tool and not a quantitative one (Heid et al. 1996). The steps for this method are outlined below.

First, mRNA is copied to cDNA by reverse transcriptase, allowing the second strand of DNA to be formed. The mix is then set up including the Taq polymerase, primers for the gene, deoxynucleotides and a buffer to resist pH change. The mix is heated to 90 degrees Celsius so that the strands separate, and then the mix is cooled to around 50 degrees. The primers take effect and bind to the sequence with which they fit. The temperature is then raised again to 72 degrees so that the Taq polymerase can copy the DNA from the primers. This results in four cDNA strands from the two that were there initially, and at this point these new strands must again be denatured at the high temperature. This process is repeated until desirable results are come to. When around 30 to 40 rounds are completed, the products of the reactions are analyzed, usually using agarose gel electrophoresis (Hunt). There are other methods that are used in addition to gel electrophoresis.

Application in Wine Microbiology:

PCR is an extremely useful tool when it comes to wine microbiology. DNA amplification methods have become a quick and reliable technique for the identification of organisms. Winemaking is no exception to this, as it is crucial that the winemaker know what organisms are contaminating a spoiled wine or why certain flavors are present. Historically, people have used plating techniques to try and isolate bacteria and yeasts to identify them, but this process is both laborious and can result in various errors. Quantitative PCR is a wonderful tool to use when identifying microorganisms in a wine medium. Firstly, many microbiological populations do not respond to standard enrichment and plating procedures, making it seem as if they are simply not present in wine when they really are.  Additionally, DNA identification takes much less time and labor, which means money.  The DNA can also be stored for long periods of time using quantitative PCR, and can be analyzed later (Phister et al. 2007).

Quantitative PCR has been developed to identify Oenococcus oeni, lactic acid bacteria, acetic acid bacteria, Dekkera, Bruxellensis, S. Cerevisiae, and Zygosaccharomyces species. Quantitative PCR has proven superior to other methods used in the past because it can quantify the population size instead of simply being a qualitative tool. Furthermore, the method is performed very rapidly and is able to simultaneously examine multiple species at one time. This method is done easily in the lab and the Taq polymerase used makes it so that it is not necessary to keep adding polymerase.

References:

  • Heid, Christian A.; Stevens, Junko; Livak, Kenneth J.; Williams, P. Mickey.  “Real Time Quantitative PCR.”  Genome Research.  6, 986-994. 1996.
  • Hunt, Margaret.  “Real Time PCR.”  Microbiology and Immunology On-Line.  University of South Carolina School of Medicine. http://pathmicro.med.sc.edu/pcr/realtime-home.htm
  • Phister, Trevor G.; Rawsthorne, Helen; Joseph, C.M. Lucy; Mills, David A.  “Real-Time PCR Assay for Detection and Enumeration of Hanseniaspora species from Wine and Juice.”  Am. J. Enol. Vitic.  58:2.  2007.

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