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17th January 2022
As someone who has worked on this machine from the early 2000’s, I never would have imagined that post-2019 ‘Real Time PCR’ would become a household name, but that’s exactly what Real Time is. The world has changed forever after 2019 & this word Real time PCR would ever be etched in the memory of survivors; for some as its results were & are & will be a relief, for others its results give a sinking feeling & will continue to; but for all those who have run the experiments on real time let me say its an enriching experience as we come face to face with what our genes do, behave or what infections we have. The following section is a very thin informative tapestry of the technique which is often called the ‘Queen of Gene Expression’.
The invention of polymerase chain reaction (PCR) technology by Kary Mullis in 1984 was the impetus in giving birth to real-time PCR in the Mid 1990s. Detection and expression analysis of genes in real-time has revolutionized the 21st-century biological sciences due to its tremendous application in quantitative genotyping, genetic variation of inter and intra organisms, early diagnosis of disease, copy number detection, SNP genotyping are to name a few of its applications. It distinguishes itself from other methods available for gene expression in terms of accuracy, sensitivity, and fast results. The technology has established itself as the golden standard for medium-throughput gene expression analysis. The assay realizes the inherent quantitative potential of the PCR, making it a quantitative as well as a qualitative assay.
The term ‘quantitative PCR’ is considered an oxymoron due to the non-linearity of the relationship between amplicon yield and starting copy number. However, using in-depth statistics these bottlenecks can be evaded giving us pure expression results. There are two main Molecular biology techniques used to work around the problematic kinetics of PCR in order to obtain accurate quantitative data, one is competitive quantitative PCR, and other is Real-Time PCR. Amongst them, Real-Time PCR is the clear winner due to the generation of statistical outputs like the efficiency of slope, dynamic range precision, correlation coefficient, and sensitivity. All these can be gleaned out of raw real-time data via florescent signal capture.
The fluorescent assay chemistries are mainly divided into two classes; sequence-independent detection assays constituting DNA-binding dyes like SYBR Green I, EtBr, Eva green, Cyto 9. sequence-specific probe or primer binding assays like molecular beacons, TaqMan, hybridization, Eclipse probes and Amplifluor, Scorpions, LUX, BD QZyme primers.
Real Time PCR quantification assays & strategies: Quantification assays which are based on the template can be divided into DNA / cDNA and RNA assays (one step and two-step RT PCR). Based on reaction type they can be categorized into simplex and multiplex reactions. Quantification is based on the mathematical models; absolute quantification is used in copy number detection and zygosity testing and for expression related studies relative quantification is the norm, which in turn has two, models one with unit mass reference and other by using endogenous control as reference. There are two statistical methods used along with relative quantification: Livak’s method [without efficiency correction] and Pfaffl’s method [with efficiency correction].
The applications of Real time PCR in Science ranges from the genetics of up regulation and down regulation of genes, cis-transgenics, quantification of pathogenic and symbiotic association of micro-organisms with plants, animals, contamination of processed food by foreign DNA, integrated expression analyses, gene families, SNP Genotyping and allelic determination, mutation detection, confirmation of microarray experiments, sanger sequencing results & ratification of Next Generation Sequencing libraries.
In today’s world with multiple pandemics and endemics ranging from different parts of the world Real-time PCR has become a benchmark to decide patients fate and draws the golden line of application of Science in life and death situations.