PCR FUNDAMENTALS

By Kory Davis
QC & Propagation Specialist
The Brewing Science Institute

PCR, Polymerase chain reaction, is a molecular testing technique that amplifies genetic material so that it can be detected.  In order to test for a specific genetic sequence, the user must first have an extensive theoretical knowledge about the subject.  This theoretical knowledge encompasses the basic building blocks of life, DNA, all the way up to how those building blocks orchestrate living mechanisms.

An image of rotating DNADNA is a double helix comprised of three chemical components: phosphate, deoxyribose, and four nitrogenous bases.  The phosphate group and the deoxyribose constitute the “backbone” while the nitrogenous bases connect two “backbones” together.  The four nitrogenous bases are adenine (A), guanine (G), cytosine (C), and thymine (T).  Each nucleotide always pairs with its corresponding base due to structural requirements.  In DNA, adenine always pairs with thymine and guanine always pairs with cytosine (Peter J. Russell, 2010).

DNA is essentially a biological codex that gives rise to life.  Once a specific genetic sequence is determined, PCR can be utilized.  To begin PCR, a primer needs to be developed.  Primers are the antithesis to a pre-determined genetic sequence and are the starting point of PCR.

An image of DNA coding from National Human Genome Research Institute.Once the primer is developed, the PCR reaction can take place.  Using a synthesized primer, Taq polymerase, oligonucleotides, and magnesium chloride the researcher can put the organism of choice through a thermocycle reaction.  This reaction begins by heating the DNA to range of 94⁰C-98⁰C, which denatures the hydrogen bonds between the nucleotide bases.  After denaturing the DNA, the primer anneals or “sticks” to the DNA at a temperature between 50⁰C-68⁰C.  After the primer has annealed the temperature rises to 70⁰C-74⁰C and Taq polymerase attaches to the sequence to begin amplification.  This process repeats ~30-40 times, amplifying the sequence of choice to a quantity that is detectable for modern methods.  This amplified DNA can then be detected by various end user methods (Peter J. Russell, 2010).

In the brewing industry, PCR is a useful tool for the rapid detection of contaminant organisms.  Mainstream organisms of interest are Saccharomyces cerevisiae var diastaticus, Lactobacillus/Pediococcus, and Brettanomyces.  At BSI we offer PCR contamination testing for the organisms listed above.  For more information please visit our website at, https://brewingscience.com/contamination-testing/

References

Deoxyribonucleic Acid (DNA). (n.d.). Retrieved from https://www.genome.gov/genetics-glossary/Deoxyribonucleic-Acid

Pearson Education. (2010). IGenetics: a molecular approach.

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