GENE EXPRESSION AND PHYSIOLOGY

Real-time Polymerase Chain Reaction

What it is:

Monitoring how many copies of DNA are produced by PCR

Real-time PCR is a form of polymerase chain reaction where the amplification of a DNA target is monitored directly in the reaction tube. As a reminder, PCR is a technique for amplifying (making millions of copies of) a specific DNA sequence. In “conventional” or end-point PCR, the only way to confirm the presence of the amplified DNA is to run an agarose gel (DNA gel electrophoresis) or to carry out another assay at the end of the PCR. In real-time PCR we can literally just monitor the process inside the tube through a camera or light sensor built into the PCR machine. Real-time PCR instruments can follow the doubling of target DNA during each PCR cycle by directly measuring light emitted by the PCR product as it accumulates and activates a fluorescent DNA dye. The amount of fluorescence increases as DNA gets amplified inside the tube. As each new copy of DNA becomes fluorescently labeled, it allows the user to keep track of how much DNA is produced. The higher the initial amount of target DNA in the sample, the faster the fluorescence will increase–giving us an idea of how much target DNA was in the sample to begin with! Because it allows us to estimate the starting amount of target DNA, real-time PCR is also known as quantitative PCR (qPCR).

The Debate

Why not use traditional PCR?

The greatest advantage of real-time PCR is that it can be used to produce quantitative data of gene expression. For example, if researchers want to know when a certain gene is up- or down-regulated, qPCR can help compare cDNA samples for changes in the expression of that gene. What’s more, qPCR can be done with very little starting material, and its efficiency can be precisely quantified. More generally, a practical benefit of qPCR is that it does not require a second step, such as gel electrophoresis, for detecting the copied DNA and finding out when reactions worked or failed. Less sample handling means less room for procedural errors.

However, there are disadvantages to qPCR, such as requiring expensive equipment, reagents, and more complicated data analysis than conventional PCR. There are many day-to-day situations when conventional PCR is sufficient, but when precise quantification of a nucleic acid sample is needed, qPCR is the preferred assay.

How it is used:

A molecular assay to dye for

The first step in designing a real-time PCR experiment is to choose the correct fluorescent dye for your specific application. There are two main options to choose from: intercalating dyes (e.g., SYBR® green) and probe-based detection (e.g., Taqman®). Both are designed to generate fluorescence during the PCR; intercalating dyes become more intensely fluorescent when they encounter double-stranded DNA, and probes are tethered to fluorescent dyes that are only released as a specific PCR product is made. Because probes are specific to the target DNA they directly confirm amplification of the desired target, whereas intercalating dyes fluoresce when bound to any double-stranded DNA, including non-specific primer dimers and off-target amplification.

Molecular diagnosis and gene expression studies

One key application of qPCR is molecular diagnostics for diseases. qPCR is a powerful tool to identify infectious microorganisms and viruses (e.g. hepatitis B, HIV, zika). qPCR is also used to validate the findings of gene expression studies (such as those involving DNA microarrays and GWAS). This often involves reverse transcription qPCR (RT-qPCR), which starts by converting mRNA to copy-DNA (cDNA) before that cDNA can be amplified by PCR.

The future:

Detecting microorganisms in food and profiling cancers

Applied uses of qPCR focused on using it to detect and quantify microorganisms, for example in food and environmental samples (e.g., air, water.). This is preferable to traditional microbiology culture methods because qPCR is much faster and more sensitive to detect small amounts of microorganisms. Understanding the microorganisms present in food is important to prevent food-borne illness and improve public health. Newer, more portable real-time PCR devices will help make that possible to do directly in the field, without the need to run gels and interpret bands!

Another growing use of qPCR is profiling cancerous tumors. qPCR can be used to understand the genetic profile or molecular markers in a certain tumor, and develop a more effective treatment plan for every individual case.

Questions – Coming Soon!