The polymerase chain reaction (PCR) is a powerful molecular biology technology that is used to recognize a specific DNA sequence and amplify a large number of precise copies of it efficiently. This enables a variety of applications, including forensic and clinical diagnostics. There have been numerous PCR derivatives since the development of this method, including quantitative (qPCR) and reverse transcription polymerase chain reaction (RT-PCR), respectively. A PCR reaction, in general, has three phases: exponential, linear, and plateau. In the exponential phase, the product is precisely doubled at each cycle, and in the linear phase, reaction components begin to deplete at varying rates due to the different reaction kinetics in each reaction tube or well; thus, each sample will plateau at a different point. Overall, conventional PCR quantifies data from the linear and plateau phases, which means it is only semi-quantitative at most, allowing only a tenfold change in gene expression to be detected. While qPCR measures the reaction at the exponential phase, it enables the detection of a twofold change in gene expression.
As the reaction proceeds, qPCR enables the identification and quantification of the target DNA; various fluorescent chemistries relate PCR production concentration to fluorescence intensity. The threshold is the level of fluorescence above the baseline, which corresponds to the three standard deviations above the mean baseline values, within the exponential phase. Reactions are defined by the PCR cycle at which amplification of a PCR product is first detected. The baseline in the amplification plot is the average background. The threshold cycle (also known as the CT value) is the cycle in which the fluorescent signal exceeds the background and crosses the threshold. The lower the CT, the more target DNA is present in the sample and the sooner the fluorescence crosses the threshold. Absolute and relative quantification can be utilized for qPCR normalization.
This technique is used to amplify RNA targets for RT-PCR. The enzyme reverse transcriptase uses RNA as a template to create complementary DNA (cDNA). Exponential amplification is performed using the cDNA template. Only sequence-specific primers may be used in one-step RT-PCR, which combines the RT reaction and PCR reaction in the same tube or well. In two-step RT-PCR, the generated cDNA is moved into a second tube for PCR.
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