By Ayisha Malik, EMEA
PCR is the basis of many research, diagnostic, forensic, and testing procedures.
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.
Types of PCR techniques
PCR mimics what happens in a cell when DNA is replicated; a biochemical technique that quickly amplifies DNA quantity. 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, the sample is heated until the DNA double helix strands separate. The temperature is then lowered to optimise primer binding and then again raised to facilitate the elongation of new DNA molecules; doubling the quantity of the DNA of interest with each cycle.
Quantitative PCR (qPCR) works by using fluorescent probes; 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 concentration. Compact and fast cycling machines such as the PCRMax® Eco48 Real-Time qPCR System can offer reliable results in less than 40 minutes.
qPCR has been also been adapted for amplification of RNA molecules; a crucial development in the detection of retroviruses, such as HIV, and analysis of mRNA transcripts associated with cancers. Reverse transcription polymerase chain reaction (RT-PCR) is a two-step process; the RNA is first converted to its complementary DNA (cDNA) by the reverse transcriptase enzyme 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 for specialist medical applications. A sample of DNA is divided at the start of the reaction, to ensure no more than one molecule in present in a well. Following amplification, absolute quantification can be determined by a simple calculation of the positive and negative wells. The level of precision can help doctors understands the copy number for genes involved in certain cancers.
Multiplex PCR (mPCR)
Often used in diagnostic laboratories, mPCR simultaneously uses multiple pairs of primers to amplify several regions of the same nucleic acid at the same time, making it possible to diagnose several diseases with a single test. The PCRmax® Eco 48 thermal cycler allows for simple multiplex analysis with up to four dyes at once.
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