PCR vs qPCR aka endpoint or real-time

Probably one of the hottest methods right now in the world of science is PCR (polymerase chain reaction). What started in 1983 as a simple method to amplify DNA now has many different variations and endless application possibilities. As of today, PCR reached the “golden standard” status and is the most applied method in molecular diagnostics with hard-to-beat accuracy and reliability. PCR is routinely applied in infectious disease diagnostics, but also genetic, oncology, prenatal screenings. We doubt that the PCR inventor would have ever guessed that this would become the method eventually transferring the whole healthcare industry. Today, using the same PCR reaction we are basically able to detect anything within minutes, which is a powerful and irreplaceable tool in the hands of medical professionals.

The most popular variations are probably the conventional PCR aka endpoint PCR and qPCR also known as real-time PCR or RT-PCR. To avoid any confusion, we are going with endpoint PCR and qPCR.

Both of these variations have a quite similar general mechanism. First, you need purified DNA (not mandatory, but recommended). The larger part of the PCR reaction occurs in three main steps, repeated around 20-40 times.

1. Denaturing of the DNA chains – DNA is heated to ~95 °C where the DNA double helix is separated into two individual DNA strands.
2. Annealing – the temperature is lowered to about 55°C so that the primers can attach to the individual strands.
3. Elongation (also used as an extension) – the temperature is once again raised to ~72 °C so that DNA polymerase can create a complementary strand to each DNA strand we had after the first step.

Differences between endpoint PCR and qPCR

There are of course differences between endpoint PCR and qPCR. Endpoint PCR is typically used for qualitative purposes to detect genomic fragments (if present or not). The results are analyzed at the end after all the PCR cycles have been completed. Agarose gel electrophoresis or hybridization-based microchip analysis is often applied for result analysis because it allows visualization of the results. There are also many variations to endpoint PCR – standard or fast cycling, regular or hot-start, etc. – which you have to pay attention to when choosing the right product for your experiment. (More information about that below.)

Then we have qPCR, which is mostly used for quantitative purposes to detect, quantify and characterize genomic fragments. Results are collected during the reaction in real-time with the help of fluorescent dyes or reporter probes. Compared with endpoint PCR, qPCR is less time-consuming, doesn’t require any messing around with agarose gel, and has a higher resolution. Like endpoint PCR it has many variations to consider: dye or probe, standard or fast, etc.

What to choose, PCR or qPCR?

If reading about the different mechanisms didn’t give you any clues as to which one you should use for your experiment, then here are a few examples that might make the choice a bit easier. You could use endpoint PCR if you are interested in cloning, genotyping, sequencing, finding if a certain disease is present in a patient, if a man is indeed the father, whether you are related to mummies or if a suspect did the crime.

If you are more interested in gene expression analysis, infectious diseases, cancer, food, and water safety (including GMOs) you should try qPCR. Of course, in most cases it is possible to use both, so the choice is really up to you.

Whether you have decided to go with endpoint PCR or qPCR you can find all you need in Solis BioDyne’s product list, which also gives you the possibility to try out different variations of endpoint PCR and qPCR and choose what suits you best.