What is PCR?
PCR, Polymerase Chain reaction is a lab technique developed by Kary Mullis in the 1980s, it is used to create a large amount of DNA from a small amount of DNA. PCR is usually a step used in other processes, for example DNA sequencing, detection of pathogens, and gel electrophoresis. PCR is an essential diagnostic technique which is frequently used in fields such as virology, microbiology, parasitology, mycology and dentistry.
What is qPCR (Real-Time PCR)?
PCR had limitations as it was not quantitative, it only revealed the presence or absence of a target. The development of qPCR, also known as Real-Time PCR or RT-PCR, has changed that. qPCR allows quantitative analysis via the addition of a fluorescent dye. Software is used to detect and analyse the signal generated when fluorescence is emitted from the sample. The amplification of the signal is in direct proportion to the amount of PCR product therefore, results are quantitative. qPCR generates faster and more accurate results as they are available in real time as the experiment progresses, it is a useful tool in clinical diagnostics as well as research.
What is Reverse-transcription PCR?
qPCR comes into its own with the ability to measure gene expression. In gene expression, mRNA is synthesised when a section of DNA is read, the mRNA is then used to produce proteins. In the process of reverse transcription, reverse transcriptase can be used to create complementary DNA (cDNA) which will be copies of the RNA being studied. qPCR is then carried out on the cDNA to give accurate measurements of the amount produced. RNA can be used in both PCR and qPCR, if RNA is used in PCR it is known as RT-PCR, if RNA is used in qPCR it is known as qRT-PCR.
Applications and uses of PCR and qPCR
PCR is a relatively simple process which will detect presence or absence of DNA. qPCR gives real time data measuring reaction rates providing information about the quantity of DNA present in the sample at any given time, essentially, it is capable of measuring the rate of gene expression. In molecular biology this facilitates an understanding of protein synthesis and therefore biological pathways in health and disease. Real-Time or qPCR is useful for detection of gene expression, presence and amount of pathogens and RNA as well as for validation of DNA microarray results and for environmental applications. qPCR has the advantage of high throughput and a broader dynamic range as it can detect wide ranging levels of expression within the same well. Because of its multiplexing capabilities where multiple targets can be amplified within the same experiment, high throughput and rapid results, it can be more economical to run.
Equipment for use with PCR and qPCR
qPCR is the best choice for amplification, characterization, quantification and analysis of nucleic acids. Used with fluorescent dyes, It is perfect for use with optical technology, therefore good quality, clear microplates and films must be used. View our qPCR product range. PCR is also used to amplify and detect short sequences of DNA for subsequent analysis e.g. gel electrophoresis. Microplates and films do not need to be clear as PCR is not used in optical applications, as ever, it is advisable to choose good quality microplates that will be reliable and durable in the long term, view examples here. For further advice and support, the iST team is available for you, please contact us for information.