PCR & Real-Time PCR
PCR & Real-Time PCR
The difference between PCR an qPCR
Polymerase chain reaction (PCR) is a widely used molecular biology technique to amplify DNA and RNA sequences. To reach a detectable level, multiple identical copies of a DNA template are created. A PCR requires a thermocycler, a DNA polymerase, Nucleotides, Primers and a DNA template. Once the ingredients for the PCR have been mixed together, the amplification of the template can start using a thermocycler. The thermocycler will perform and repeat a cycle of different temperatures. There are three main stages during each PCR cycle:
Denaturation: The double stranded DNA is heated to separate it into two single strands
Annealing: The temperature is lowered to enable the DNA primers to attach (anneal) to the DNA template
Extending/Elongation: The temperature is raised to the optimal working temperature of the PCR enzyme, allowing the enzyme to create and the new strand of DNA, using the Nucleotides to build the strand.
After each PCR cycle, the number of DNA strands are doubled.
The exact temperature and time of each stage depend on the primer sequences, DNA fragment lenght and DNA Polymerase being used. In general the temperature of the denaturing stage is 94-95°C, annealing stage is between 50-65°C and extending stage is around 72°C.
Quantitative PCR (qPCR) will amplify a specific DNA template segment based upon the PCR primers as well. However, it will also allow measurement of the DNA template concentrations. The amplification of the DNA can be measured throughout each amplification cycle, due to the presence of an DNA binding dye, for example SYBR® Green. SYBR® binds only to double stranded DNA and once bound changes its structure releasing a fluorescent signal.
Since each PCR cycle accumulates the amount of double stranded PCR product in the reaction vessel, the signal will increase with each cycle parallel with the DNA concentration. Next to the DNA sample(s), a series of DNA standards will be amplified as well. These standard will help forming a calibration curve, which enables scientist to plot and determine the concentrations of their DNA samples.
And what about RT-PCR and Real-Time PCR?
qPCR and Real-time PCR are the same techniques, and are explained in the paragraph above. But RT-PCR means something different. A RT-PCR is short for Reverse Transcriptase PCR. This method allows the use of RNA as a template. With the use of an enzyme called reverse transcriptase, RNA is translated into complementary DNA (cDNA). Most commonly, RT-PCR serves as a first step in qPCR, which quantifies RNA transcripts in a biological sample. It is then called RT-qPCR.
To make this even more complex, the abbreviation frequently used for Real-Time PCR is RT-PCR.