What is qPCR and how is it used in practice?
PCR (polymerase chain reaction) transformed biological research. During the COVID pandemic, more people were talking about PCR than ever before. Quantitative real-time PCR (qPCR) is a powerful technique for showing not only whether a nucleic acid sequence is or is not present in a sample, but for determining exactly how many copies of that sequence are present in the sample.
So what is it? How is it used? And where do you turn when your reactions aren’t working how you expected them to? This is part I of a two-part blog series. In Part I, let’s define some terms:
What is PCR: Polymerase chain reaction
The development of the PCR technique to amplify specific pieces of DNA won its inventor, Kary B. Mullis, the 1993 Nobel Prize in Chemistry. PCR takes advantage of the double-strandedness of DNA. The steps in PCR are: 1) DNA in a sample is sequentially heated to separate the strands, 2) cooled to allow two primers (each specifically designed to bind to one strand bracketing the sequence to be amplified, to anneal to the separated DNA strands) and 3) incubated with a DNA polymerase which binds to the annealed primers and synthesizes the complementary DNA strands.
At the end of each cycle, one piece of double-stranded DNA has been copied into two double-stranded pieces. Over subsequent PCR cycles, each copy is itself copied, so the number of copies grows exponentially.
Because PCR amplifies the target sequence exponentially, if you start with a single piece of DNA, after 20 cycles of PCR, there will be over a million copies of the target sequence. PCR can be used to make enough copies to visualize on a gel, construct recombinant plasmids, use as probes in new experiments, use in a diagnostic test, or in other downstream applications.
What is qPCR: Real-time PCR
Let’s clear the air. There can be confusion around the term RT-PCR, which stands for “reverse transcription PCR.” Real-time PCR is commonly known as qPCR (the “q” stands for quantitative”), where the accumulation of PCR products is followed in “real time” by incorporating fluorophores into the product and monitoring the fluorescence of the reaction.
Ct value: cycle threshold value
When watching fluorescence increase as PCR products accumulate in real time, initially the fluorescence signal will be too low for the detector to pick up. Scientists select a threshold value above which the fluorescence signal can be confidently detected. To build a standard curve, they see how many cycles it takes for a reaction with a known starting amount of target sequence to reach the fluorescence threshold value. That cycle number is Ct.
qPCR: quantitative real-time PCR
qPCR allows quantitation of the amount of target in a sample. Comparing the increase in fluorescence signal of the sample to a standard curve made by amplifying known starting amounts of the target sequence, the amount of target in an unknown can be calculated.