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By Ayisha Malik, EMEA
Everyone has been touched by polymerase chain reaction (PCR) technology in one way or another. It is an innovative biochemical tool that has propelled healthcare and research into the 21st century. The simple yet magnificent process was only invented four decades ago but has since revolutionised molecular biology. Today it forms the basis of many research, diagnostic, forensic, and testing procedures.
According to research , the global PCR market, valued at over US $7,027 million in 2016, is estimated to reach $10,776 million by 2023; registering a compound annual growth rate (CAGR) of 6.2%. While North America is currently the highest contributor, a large growth is expected in the Asia-Pacific market over the next few years.
As PCR is adopted into different facets of science and industry, there is ongoing efforts to develop advanced PCR systems, with enhanced capabilities, such as higher sensitivity and specificity. Other major advances relate to shorter PCR cycling time, higher productivity, and automated workflows. These advancements, without a doubt contribute towards market growth. Additionally, the increase in the global geriatric population, high prevalence of infectious diseases, rise in number of diagnostic centres and hospitals, technological advancements, and increase in awareness and acceptance of personalised medicine are also attributed towards driving the PCR market size. Increase in R&D funding and the emergence of new untapped markets in developing regions are also expected to provide new avenues for the growth of PCR technologies market size in the near future.
DNA replication is the process that governs repair and reproduction mechanisms in all forms of life. With this fundamental biochemical mechanism at its core, the concept of PCR was created. It started to take shape in the 1960’s, when a heat resistant DNA polymerase enzyme was discovered in the extremophiles living in the superheated waters of Yellowstone’s Mushroom Spring. Although Kjell Kleppe, the Norwegian researcher working with Gobind Kohrana, outlined the first enzymatic assay to replicate short strands of DNA in vitro in 1971, Kary Mullins is generally credited with the invention of PCR.
In 1983, he summarised the procedure, "beginning with a single molecule of genetic DNA, PCR can generate 100 billion similar molecules in an afternoon. The reaction is easy to execute. It requires no more than a test tube, a few simple reagents, and a source of heat.”
Seven years after he first published his findings, in 1993, Mullis was awarded the Nobel Prize in Chemistry for his invention.
PCR techniques offer absolute detection and quantification of DNA and RNA that are increasingly being used in life science fields such as genetics, molecular biology, biotechnology, and drug discovery. Moreover, these technologies can also be used for criminal investigations, parental identity determination, industrial forensics analysis and of course clinical diagnostics. PCR technologies too have diversified since its inception, with varied forms, offering added benefits that suit the needs of its multiple applications.
PCR mimics what happens in a cell when DNA is replicated prior to cell division. It is a biochemical technique that quickly amplifies DNA segments of interest to generate millions of copies. For the reaction, a mixture of DNA, primers, heat-resistant DNA polymerase, nucleotides, along with suitable buffers and salts are added to a tube and placed in a thermal cycler.
To start the process, a melting temperature (Tm), upwards of 90°C, is applied to the mixture for the strands of the DNA double helix to separate. The temperature is then lowered for the primers to anneal to the newly separated single strands of DNA; an excess of primers facilitates primer-DNA binding over DNA strands reannealing. Once the primers-DNA complexes have formed, the temperature is raised but kept below Tm for the elongation process. DNA polymerase starts to incorporate the free nucleotides into complimentary DNA strands, thereby doubling the quantity of the DNA of interest. This entire process is repeated in a cyclic fashion, doubling the amount of DNA each time.
One key development in PCR technology has been the invention of real-time or quantitative PCR (qPCR). This method works by using fluorescence labelled probes and monitoring the fluorescence after each cycle to determine PCR rate in real time. The intensity of the signal corresponds to the amount of DNA amplification. Furthermore, the number of cycles at which the fluorescence is first detected, can be used to calculate the initial number of DNA molecules present in the sample. Quantitative PCR has been crucial in early diagnosis of life-threatening diseases as well as for the determination of microbial load in the prognosis of an active infection. Compact and fast cycling machines such as the PCRmax® Eco 48 can offer reliable results in less than 40 minutes.
The qPCR process has also been adapted for the amplification of RNA. This is a crucial development for the detection of retroviruses such as HIV and the analysis of mRNA transcripts associated with many cancers. Reverse transcription polymerase chain reaction (RT-PCR) is a two-step process used to detect and quantify RNA molecules in a sample. The RNA is first converted to its corresponding complementary DNA (cDNA) by the enzyme reverse transcriptase and then amplified using standard PCR. Accurate RT-PCR is also a crucial step involved in vaccine research and development.
Digital PCR offers an enhanced level of precision and is now making a comeback, mainly for specialist applications. It was invented in the early 1990’s but as qPCR took centre stage, digital PCR had to take a step back. The technique works by dividing the initial DNA sample into wells, with one or zero molecules in each. Following amplification, absolute quantification can be determined by a simple calculation of the positive and negative wells.
The level of precision offered by digital PCR is not necessary for most clinical applications. It is, however, useful when we need to know exact quantification of a target; for example, determining the copy number of genes involved in cancers that are affected by the presence of multiple copies of the same allele. Knowing this copy number can change the treatment plan, personalising it for each patient.
Multiplex PCR (mPCR) is often used in diagnostic laboratories to increase the diagnostic capacity of PCR. In mPCR, different pairs of primers are simultaneously used to amplify multiple regions of the same nucleic acid sample. The main advantage of this technique is that it makes it possible to diagnose several diseases with a single test; without the need for multiple separate reactions. The PCRmax® Eco 48 thermal cycler allows for simple multiplex analysis with up to four dyes at once.
As they say the best ideas are often the simplest and PCR is a true testament to that. Leveraging the basic DNA replication mechanism, scientists have developed a process that is now routinely applied to achieve accurate disease diagnosis, personalised therapies and faster treatments. Additionally, PCR technology has also revolutionised forensics, quality control and scientific research. As this technology evolves further, it is likely to become more ingrained into our modern lives; offering solution to problems that we currently have no answers for.