The Polymerase Chain Reaction (PCR) In Food Science

The Polymerase Chain Reaction (PCR) is a remarkable technique developed by Kary Mullis in the 1980s (more specifically 1985) for the California Biotech Company, Cetus. It is reasoned to be one of the most important inventions of the 20th Century in molecular biology. Kary Mullis was awarded the Nobel Prize jointly with Michael Smith for this work in 1993. The intention of the method is to produce large copies of a single gene which can be used for identification purposes. The production of large quantities of a specific DNA piece is termed amplification.

The technique is used by a number of organizations to identify genes. Food adulteration has been on the rise for a number of years. Consumers quite understandably start to lose faith in food standards organizations when sources of food are adulterated or contaminated. Meat is no exception! Meat foods have been adulterated with cheaper questionable sources because of economics, of partially or even completing replacing high-value meat with other types. It can even mean adding meats that are contaminated with drugs not normally used for food. But now for example, identifying genes from different meat sources is possible as a way of checking for adulteration. The presence of horsemeat in ‘beefburgers’ and other processed foods was identified using PCR (Meira et al., 2017).

A number of variants of PCR occur, some of which are discussed below with others elsewhere on this web-site. The method can be used both qualitatively and quantitatively. We’ve also discussed the method in the context of biotechnology. Here, it’s worth going into some detail about the method itself!

The Methodology

The process of DNA production this way is an in-vitro (test tube) method. 

PCR is one of the modern methods for identifying bacteria species and is especially useful in establishing organisms causing food poisoning for example. The technique has many forensic applications and is now routinely used in cloning and medical diagnostics.

In the process of PCR, an enzyme called DNA polymerase is added to a mixture of DNA bases. Under the right conditions, the enzyme will catalyse the production of many millions of copies of a single gene even from a single starting strand of DNA. 

The DNA polymerase is highly thermostable to withstand the temperature changes that take place during the process.  

PCR is the starting point or template for DNA sequencing.

The method requires the following:-

• DNA template
• oligonuceotide primers 
• Taq polymerase
• deoxynucleoside triphosphates (dNTPs)
• buffer solutions
• divalent cations such as magnesium ions 

The Taq DNA polymerase is perhaps the special catalyst in this method. It is highly thermostable and was isolated from a thermophilic bacterium called Thermuys aquaticus which is found in hot springs. This polymerase has a temperature optimum of 72ºC and can survive exposure to temperature w which denature other enzymes. It can survive to 96ºC and so will remain active after each of the denaturation steps that it is put through.

In a test tube, DNA is heated to between 92 and 98°C. At these temperatures DNA is denatured and separates from its normal two strand structure into single strands. The DNA is then cooled to between 50 and 65°C. DNA primers are added which bind to target DNA sequences which are of interest to the researcher. Complementary primers are added. These are complementary to the target sequences at the the two ends of the region of DNA to be amplified (increased in number).

The DNA mixture is heated to between 70 and 80°C. The heat tolerant DNA polymerase is added which then replicates a region of DNA to be amplified. In the process, two strands are formed.

The whole cycle of heating and cooling is repeated many times to amplify the production of the target region of DNA. A PCR machine called a thermocycler has been developed which performs this operation routinely.

The Design Of The Primers

When designing primers for PCR (Polymerase Chain Reaction) amplifications, several key features must be considered to ensure specificity, efficiency, and accuracy. The most important design features are:-

1. Length:

   • Typically 18–25 nucleotides long — long enough to be specific to the target sequence but short enough for efficient binding and extension.

2. Melting Temperature (Tm):

   • The Tm if you don’t know already refers to the temperature at which half of the DNA primer molecules are bound to their complementary DNA sequence and half are unbound — essentially, it’s the temperature at which the DNA duplex is halfway “melted.” It’s a crucial value in PCR because it helps set the correct annealing temperature during the cycles.
   • The Tm should generally be between 50–65°C.
   • Forward and reverse primers should have similar Tm values (within 3°C of each other) to ensure they anneal at the same temperature during PCR.
   • You can estimate empirically the Tm of a primer based n its nucleotide composition. For primers of between 14 and 20 nucleotides long we use the Wallace rule.

                 This is: Tm = 2(A+T) + 4(G+C).

                       Where:

1. • • A, T, G, and C are the counts of each nucleotide in the primer sequence.
     • A and T pairs contribute 2°C each to the Tm.
     • G and C pairs contribute 4°C each since GC pairs form three hydrogen bonds, making them more thermally stable.
     • For example, a primer has the sequence 5’-AGCTTGACCGTAAGT-3’.

       1. Count the nucleotides:

          • A = 4
          • T = 3
          • G = 4
          • C = 4

       2. Apply the formula:

       $$Tm=2(4+3)+4(4+4)=2(7)+4(8)=14+32=46°C$$

     • For longer or more complex primers, you might use more precise software or formulas like the nearest-neighbor thermodynamic model, but the Wallace rule is a fast way to get a rough estimate.

   • Knowing the melting temperature (Tm) of primers is essential for setting the annealing temperature (Ta) in PCR because the annealing step is when primers bind to their complementary sequences on the target DNA — a crucial moment for successful amplification.

     How Tm guides Ta selection:

     • The annealing temperature (Ta) is usually set about 3–5°C below the Tm of the primers.
     • This ensures the primers can bind efficiently without causing nonspecific binding or primer-dimer formation.

     Why not use Tm directly?

     • If Ta is too high: Primers may fail to bind at all, reducing or preventing amplification.
     • If Ta is too low: Primers may bind nonspecifically to partially matching sequences, leading to off-target products.

     Formula for estimating Ta:

     A commonly used formula is:

     $$Ta=Tm-5°C$$

     For example:

     • If primer no. 1 and primer no. 2 have a Tm of 58°C, the Ta might be set at 53°C.

     In practice, many researchers fine-tune the Ta by running a gradient PCR — testing a range of temperatures to find the one that gives the cleanest, most specific bands on a gel.

2. GC Content:

   • Ideally 40–60% GC content for stable binding.
   • A GC clamp (1–3 G or C bases at the 3′ end) can help improve binding stability without causing mispriming.

3. 3′ End Specificity:

   • The 3′ end of the primer is crucial for polymerase extension, so it must match the target sequence exactly.
   • Avoid runs of 3 or more G or C bases at the 3′ end, as they may cause nonspecific binding or primer-dimer formation.

4. Primer-Dimer and Hairpin Avoidance:

   • Primers should be designed to minimize complementarity between themselves (to avoid primer-dimers) and within themselves (to prevent hairpins).
   • Use tools like primer design software to check for these issues.

5. No Secondary Binding Sites:

   • Primers should be specific to the target sequence, with minimal chance of binding to non-target regions.

6. Product Size:

   • Ensure the primers flank the region of interest, producing an amplicon of the desired size (usually 100–1000 bp for standard PCR).

7. Balanced Base Composition:

   • Avoid long runs of a single nucleotide (e.g., AAAAA) or highly repetitive sequences, which can lead to mispriming.

Using The Thermocycler

In PCR, the thermocycler goes through a series of temperature changes in each cycle to allow DNA amplification. The three main steps — denaturation, annealing, and extension — happen at different temperatures:

1. Denaturation (94°C):

   • The high temperature breaks the hydrogen bonds between the complementary strands of double-stranded DNA, causing the DNA to “melt” into two single strands.
   • This step prepares the template DNA for primer binding.

2. Annealing (48°C):

   • The temperature is lowered so that primers (C1, C2 for chicken or P1, P2 for puffin) can bind (or anneal) to their complementary sequences on the single-stranded DNA.
   • The annealing temperature depends on the Tm of the primers — 48°C is likely chosen because it is 3–5°C below the Tm of the primers.

3. Extension (72°C):

   • This is the optimal temperature for Taq DNA polymerase, the enzyme that adds nucleotides to the 3’ end of the primers, synthesizing new DNA strands.
   • At 72°C, Taq polymerase works most efficiently, extending the new strand at about 1,000 bases per minute.

The cycle repeats — usually 25–35 times — doubling the DNA each time, leading to exponential amplification of the target sequences.

The Use Of Ethidium Bromide (EtBr)

Ethidium bromide (EtBr) is a dye commonly used in agarose gel electrophoresis to visualize PCR products because of its ability to bind to DNA and fluoresce under UV light. Here’s how it works:

1. Intercalation into DNA:

   • EtBr molecules insert themselves between the stacked base pairs of double-stranded DNA.
   • This intercalation process distorts the DNA slightly but doesn’t affect its migration through the gel in a significant way.

2. Fluorescence under UV light:

   • When exposed to ultraviolet (UV) light (usually around 302 nm), EtBr absorbs the UV energy and emits orange fluorescence at around 590 nm.
   • The intensity of the fluorescence corresponds to the amount of DNA present — so brighter bands indicate larger quantities of DNA.

3. Visualization of PCR products:

   • After running the PCR products through the gel, the DNA bands can be seen under a UV transilluminator.
   • This allows you to check for the expected band sizes.
   • Unexpected bands could indicate contamination, primer-dimers, or non-specific amplification.

⚠️ Safety note: EtBr is a mutagen and must be handled with care, using gloves and protective eyewear, and disposed of properly.

The Appearance of Non-Specific Bands

Quite often, non-specific bands are seen on agarose gels.

• These non-specific products likely form due to mis-annealing of primers — for example, if the primers X1 and X2 say bind to regions of another species’ DNA that share partial sequence similarity, or if there is primer-dimer formation.
• It could also result from cross-reactivity between the two sets of primers (X1/X2 and Y1/Y2), amplifying an unintended region of DNA.

Simple strategy to prevent this:

1. Increase the annealing temperature (Ta):

   • Raising the Ta (typically closer to the Tm of the primers) improves primer specificity.
   • A low Ta (like 48°C) allows for partial or mismatched annealing, so increasing Ta to around 55–60°C can reduce non-specific binding.

2. Other strategies (optional extras):

   • Optimize Mg²⁺ concentration: Too much Mg²⁺ stabilizes mismatches — lowering it can enhance specificity.
   • Touchdown PCR: Starting with a higher Ta and gradually lowering it each cycle can improve primer binding precision.

Real Time Detection Of PCR Products

Here no gels are needed at all. The method relies on the binding of a dye, SYBR Green which only binds to double stranded amplicons produced during PCR. This fluoresces and is detected in a fluorometer. Other dyes are available including EvaGreen (Meira et al., 2017).

Applications Of PCR

The technique of PCR has found its way into all sorts of industrial applications especially where identification is needed. Some techniques have been specifically developed for this purpose.

Multiplex PCR

In this method, several primer pairs with similar annealing properties are added to a PCR mixture so that several target sequences are detected simultaneously. The benefits are that it saves time and reduces the expense of detection of pathogens.

The primers used have to have the same melting temperature or it upsets the process. They cannot interact or affect each other in any way. It also means that amplified fragments of the same length cannot be detected.

One of the issues with a standard PCR method is it cannot distinguish between viable and non-viable bacteria. However there is an agent called ethidium monoazide that can separate viable bacteria from the dead.

In a number of examples, real-time PCR using RNA as the template is more appropriate because RNA is only found in viable microbes. In this case, RNA is first reverse transcribed to cDNA (copy DNA) and then used for amplification.

Some excellent examples of the application exist in the literature. One paper describes the identification of the three food pathogens, Listeria monocytogenes, Escherichia coli O157:H7 and Salmonella sp. (Kawaskai et al., 2010) in pork mince. The detection sensitivity for this method was 2.0 × 10² CFU/mL for each pathogen, and the quantification range was 10²–10⁷ CFU/mL with a high correlation coefficient. A single cell in 25 grams of spiked pork meat could be detected within 24 hours.

A study at the University of Warwick has shown multiplex PCR capable of detecting six bacterial pathogens associated with mastitis in sheep. These are Staphylococcus aureus, E. coli, M. haemolytica, Strep. agalactiae, Strep. dysgalactiae and Strep. uberis. The study is complicated by the presence of contaminants which remain to be resolved effectively.

A more extensive exploration of multiplex PCR in the context of biotechnology has been discussed elsewhere on this site.

RAPD-PCR

RAPD-PCR is an acronym for random amplified polymorphic DNA PCR. Here, a random primer of 10-mer is needed to generate a DNA profile.

The primer anneals to several places on the DNA template and is used to create a DNA profile which is used for microbe identification.

The advantages of RAPD is that pure DNA is not required, it is less labour intensive than RFLP and there is no need for DNA sequence data prior to application. The technique has been successfully applied to fingerprinting Listeria monocytogenes when there are outbreaks at a dairy.

Ribotyping

Ribotyping is a method for identifying and classifying bacteria based upon differences in ribosomal RNA (rRNA). It generates a highly reproducible and precise fingerprint that has been used to classify bacteria at the genus level to the species level.

We now have databases for Listeria based on 80 pattern types, Salmonella with 97 pattern types, Staphylococcus with 252 pattern types and Escherichia with 65 pattern types.

Other PCR methods were exploring:-

• Inverse PCR (iPCR)
• Adaptor-specific PCR
• Vectorette PCR
• Digital PCR

Adulteration of Meat

In the case of checking for adulteration of meat, the key element is to identify a gene that is species-specific. When adulteration of beef mince occurred with horse meat, food safety advisors used oligonucleotide primers specific for either beef or horse meat. They used the primers to amplify for an exon of a gene sequence that belonged specifically to that species. In adulteration studies of this kind, the design of the primers is critical. The most important design features are the following:-

• Specificity to Target DNA:

  • The primers must be designed to bind specifically to the unique sequences of a gene for beef meat and a gene for horse meat, ensuring they do not cross-react with non-target DNA. This prevents false positives or amplification of unrelated DNA.

• Similar Melting Temperatures (Tm):

  • The forward and reverse primers for each gene should have closely matched Tm values (typically between 50–65°C) to ensure they anneal efficiently at the same temperature during the PCR cycles. This helps produce clear, reliable amplification without incomplete or nonspecific products.

Having generated DNA for each gene it is a straightforward case of checking for the presence of the amplified gene using agarose gel electrophoresis. Ethidium bromide is an intercalating chemical that allows for the individual genes to be spotted on the gel when placed under UV irradiation. The gel separates the DNA fragments on the basis of size. 

References

Hu, Q., Lyu, D., Shi, X. et al. (2014). A modified molecular beacons‐based multiplex real‐time PCR assay for simultaneous detection of eight foodborne pathogens in a single reaction and its application. Foodborne Pathogens and Disease, 11, pp. 207– 214 (Article)

Kawasaki, S., Fratamico, P.M., Horikoshi, N. et al. (2010). Multiplex real‐time polymerase chain reaction assay for simultaneous detection and quantification of Salmonella species, Listeria monocytogenes, and Escherichia coli O157:H7 in ground pork samples. Foodborne Pathogens and Disease, 7, pp. 549– 554 (Article)

Meira, L., Costa, J., Villa, C., Ramos, F., Oliveira, M.B. P.P., Mafra, I. (2017) EvaGreen real-time PCR to determine horse-meat adulteration in processed foods. LWT January 75 pp. 408-416 (Article)

Russo, P., Botticella, G., Capozzi, V., Massa, S., Spano, G. & Beneduce, L. (2014). A fast, reliable, and sensitive method for detection and quantification of Listeria monocytogenes and Escherichia coli O157:H7 in ready‐to‐eat fresh‐cut products by MPN‐qPCR. BioMed Research International, 2014, 608296 (Article)