The Polymerase Chain Reaction

Fig. 1 - A strip of PCR tubes

The polymerase chain reaction (PCR) makes use of DNA’s natural ability to replicate itself in the presence of the enzyme DNA polymerase. This replication occurs just before cells divide by mitosis and involves the double stranded DNA molecule ‘unzipping’ to form two single strands. Complementary nitrogen bases then line up along each free strand to form two new double stranded molecules.

PCR – A Process That Tolerates Temperature Extremes

The polymerase chain reaction also utilises the fact that DNA will denature (divide into two strands) at high temperatures. In this way, the single strands that form in the first stage of DNA replication can be artificially prepared in readiness for the formation of complementary strands.

Although these and other vital processes occur at different temperatures, in 1985 Kary B. Mullis devised a way in which they could all occur in the same experimental vial (see figure 1) without denaturing the polymerase enzyme. He did this by using the DNA polymerase from a heat resistant strain of bacterium called Thermus aquaticus . In this way, temperatures during PCR reactions can range from around 55°C to 95 °C without denaturing the Thermus aquaticus (‘Taq’) enzyme.

The ‘ingredients’ needed in PCR include a segment of the DNA strand to be analysed, free nucleotides containing the nitrogen bases adenine, thymine, guanine and cytosine, synthesised ‘primer’ sections of DNA (base sequences that occur on either side of the desired DNA strand), Taq polymerase and a primase enzyme that anneals the primer sequences to the unzipped DNA strands.

Billions of Copies of the Desired DNA Segment in Three Hours

The PCR process commences by heating the reaction mixture to 95°C. This denatures the DNA sample to form two separate strands. The temperature of the mixture is then reduced to 60°C, ideal conditions for the primer sequences to form hydrogen bonds with their complementary sections on the free DNA strands.

Fig. 2 - The PCR Process 

Following this step, the temperature is elevated to 72°C. This is the optimum temperature for the Taq polymerase enzyme, which sets about adding complementary nucleotides to the desired segments of the DNA strands until two new double stranded DNA copies of the original segment are formed. After 30 cycles of these steps (see figure 2), which take about 3 hours to complete, up to one billion copies of the required DNA segment can be produced.

Uses for PCR in Biotechnology

The polymerase chain reaction can be used in any instance where multiple copies of a desired DNA sequence are required for analysis. Examples of this include situations where minute DNA samples from blood, tissue or bodily fluid samples found at crime scenes need to be amplified for identification using electrophoretic separation techniques, or in archaeology where small amounts of mitochondrial DNA from bone fragments or teeth can be multiplied and examined.

Other uses for PCR include the diagnosis of diseases, where unknown pathogens can be identified from their DNA. Examples of diseases that have been diagnosed in this way include AIDS, Lyme disease, middle ear infection, tuberculosis, chlamydia infection and viral meningitis. PCR techniques are often more efficient than attempts to culture and identify the various microorganisms that cause these diseases.

PCR was also instrumental in the isolation and amplification of human genes during the Human Genome Project and is used in other gene sequencing procedures. In addition, PCR is being increasingly employed in recombinant DNA techniques as a means of producing multiple copies of transgenes – genes from one organism that are inserted into the genome of another. This method is proving to be more effective than cloning the desired gene in vectors such as bacterial plasmids, as the risk of mutations is reduced.

Recent Innovations in PCR Technology

Initially, DNA segments produced in PCR were identified using gel electrophoresis, but the advent of ‘real time PCR’ has obviated the need for this in some cases by using fluorescent probes or dyes which can be detected using optical sensors. Another improvement in PCR technology has been the development of PCR cycler machines which can automatically switch between the different temperatures required in the reaction. Prior to this the PCR tubes had to be manually moved between water baths which were set at different temperatures.

The polymerase chain reaction has been described as one of the most important scientific breakthroughs to have occurred in the last hundred years and, according to Tabitha Powledge, has ‘utterly transformed the life sciences’. It has effectively made DNA readily accessible in all areas of scientific research.

References

Bethseda, MD, 1992, ‘Polymerase Chain Reaction- Xeroxing DNA’, Access Excellence Resource Centre, accessexcellence.org, accessed 4/7/2010

Dolan DNA Learning Centre, ‘Polymerase Chain Reaction’, Biology Animation Library, dnalc.org, accessed 4/7/2010

Dolan DNA Learning Centre, 'Naming PCR', Biology Animation Library, dnalc.org

, accessed 4/7/2010

National Human Genome Research Institute, 2010, ‘Polymerase Chain Reaction, PCR’, Genome.gov, accessed 5/7/2010

Powledge, T. M, ‘the Polymerase Chain Reaction’, Breakthroughs in Bioscience, faseb.org, accessed 5/7/2010