The Polymerase Chain Reaction (PCR) is a remarkable technique developed by Kary Mullis in the 1980s (more specifically 1985) for the Califirnia 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. The production of large quantities of a specific DNA piece is termed amplification.
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
- 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 poilymerase has a temperature optimum of 72 centigrade and can survice exposure to temperature w which denature other enzymes. It can survive to 96 centigrade 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.
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.
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.
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.
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 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.
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), , 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), , et al. (
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), , , , & (