Question

What is quantitative real-time PCR (qPCR)? Describe what happens during a qPCR reaction and how it is quantified.

Short answer

Answer: Quantitative real-time PCR (qPCR) is a laboratory technique used to amplify and simultaneously detect or quantify a targeted DNA molecule by monitoring the amplification process in real-time. It involves a reaction with components like DNA template, primers, dNTPs, DNA polymerase, and a fluorescent reporter. The qPCR reaction consists of three stages – denaturation, annealing, and extension – repeated for multiple cycles, resulting in exponential amplification of the target DNA. The quantification of qPCR relies on the use of a fluorescent reporter molecule that correlates the fluorescence emitted with the amount of DNA being amplified, with common methods being dye-based and probe-based. Comparing the cycle at which fluorescence signal exceeds a predefined threshold (Cq value) enables the relative or absolute quantification of the target DNA in the samples.

Step-by-step solution

Introduction of qPCR

Quantitative real-time PCR (qPCR) is a laboratory technique used to amplify and simultaneously detect or quantify a targeted DNA molecule. It is a type of polymerase chain reaction (PCR) that enables real-time monitoring of the amplification process, allowing for quick and accurate quantification of the DNA.

Components of qPCR

The main components of a qPCR assay include DNA template, primers, dNTPs, DNA polymerase, and a fluorescent reporter. The DNA template is the sample containing the DNA to be amplified. Primers are short, synthetic oligonucleotides with sequences complementary to a specific region of the target DNA and are required for the DNA polymerase to initiate the synthesis. dNTPs are the building blocks for DNA synthesis. The DNA polymerase enzyme synthesizes new DNA strands by adding dNTPs in a template-directed manner. The fluorescent reporter is crucial to measure the amplification as it happens in real-time.

Stages of qPCR reaction

A qPCR reaction typically consists of three stages – denaturation, annealing, and extension: 1. Denaturation: The DNA sample is heated to a high temperature (around 95°C) to break the hydrogen bonds between the two DNA strands, resulting in the separation of the double-stranded DNA into single strands. 2. Annealing: The reaction temperature is lowered (usually 50-65°C) to allow the primers to bind to the target DNA by forming complementary base pairs. The temperature is set to be optimal for the specific primers used. 3. Extension: The temperature is increased to the optimal temperature of the DNA polymerase (usually around 72°C). The enzyme synthesizes new DNA strands by adding dNTPs to the 3' end of the annealed primers, using the original DNA strands as a template. These three stages are repeated for multiple cycles, in which the amount of target DNA doubles with every cycle, resulting in exponential amplification of the initial template.

Quantification of qPCR

The quantification of qPCR is based on the use of a fluorescent reporter molecule, which either directly or indirectly correlates the fluorescence emitted with the amount of DNA being amplified. Two common methods are: 1. Dye-based: A double-stranded DNA-binding dye (e.g., SYBR Green) fluoresces when bound to double-stranded DNA, while being less fluorescent when free. As the amount of amplified DNA increases, so does the fluorescence signal. 2. Probe-based: This method involves the use of sequence-specific fluorescent probes (e.g., TaqMan probe) that emit fluorescence only upon hybridization with the target DNA. A quencher molecule suppresses the fluorescence of the reporter until the probe binds to its target and gets cleaved by the DNA polymerase, allowing the signal to be detected. The fluorescence signal is detected during the extension phase of each cycle, and the increase in fluorescence reflects the amount of amplicon produced. The quantification is carried out by determining the cycle at which fluorescence signal exceeds a predefined threshold (the quantification cycle or Cq value). Comparing the Cq values of samples enables the relative or absolute quantification of the target DNA in the samples.