It's all in the mix!
By: Roche Life Science
Posted: September 09, 2015 | Lab Life - Real-Time PCR
Is your qPCR master mix optimized? What makes your master mix optimized? Indeed, not all qPCR reagents are designed equally and optimization of your qPCR master mix for your specific applications is essential for the success of your assays.
Your qPCR master mix should contain all the reagents for the qPCR reaction that are not specific to the samples and assays. This includes the polymerase-type enzyme(s) (some mix will include a polymerase, some will also include a reverse transcriptase for RT-qPCR, while some will have hybrid enzymes that can use RNA as their template with a polymerase activity), an appropriate bivalent cation (most commonly Mg2+, sometimes Mn2+), dNTPs, dyes (if not performing probe-based qPCR) and a buffer that optimizes the activity and stability of the enzyme(s). Often, the buffers in your master mix contain proprietary additives and/or enhancers that may differ between various master mixes.
We will go through the essential components of your qPCR master mix, how each can be optimized for your qPCR applications, and what variations in master mixes are available for your experiments.
Master mix optimization: The enzyme
Let's start with the all-important workhorse of qPCR, the most common enzyme, the DNA polymerase. Indeed, PCR technology has come a long way since the discovery of the thermophilic bacterium Thermus aquaticus from the Lower Geyser Basin at Yellowstone National Park nearly 50 years ago1. T. aquaticus (or simply Taq, as it has come to be known) became the go-to source for thermostable enzymes.
However, we now know that heat stable is not always enough, and the ability to perform hot start PCR reactions can reduce mispriming with non-specific DNA amplification by limiting polymerase activity that occurs at room temperature or even on ice, during conventional PCR. DNA polymerases can be made hot start compatible with the addition of anti-Taq antibodies, which binds to Taq polymerase and blocks polymerase activity at annealing temperatures.
Recently, single-stranded nucleic acid (aptamer) based inhibitors are being utilized for high efficiency hot start PCR reactions. These temperature sensitive inhibitors reversibly bind to DNA polymerase at lower temperatures, and release during normal cycling temperatures. This hot start function is especially important when using automated (or robotic systems), as it means your samples are safe to sit at room temperature for up to several hours without concern for mispriming.
Ad for RT-qPCR reactions, it requires two enzymes, a reverse transcriptase to reverse transcribe mRNA to cDNA, as well as a DNA polymerase to amplify the target DNA. This has resulted in the development of chimeric thermostable polymerases with dual reverse transcriptase and DNA polymerase activities, as well as modulated proofreading activity to enhance fidelity of the reverse transcriptase. Indeed, these designer polymerases for single-enzyme RT-qPCR are ideal for a simplified setup with robust performance.
Master mix optimization: The dNTPs
The next most essential component to consider for optimization of your qPCR master mix is the dNTPs. You might think of these nucleotides as a less dynamic aspect of your qPCR reaction, but there are a few key elements to understand with respect to optimization. Indeed, the most common source of contamination in PCR reactions is usually the carry-over of previously amplified DNA. One convenient strategy employed to combat this is via the inclusion of dUTP and uracil-DNA-glycosylase (abbreviated UNG) to your reaction.
This method prevents contamination with carry-over amplicons by degrading PCR products from prior amplifications without degrading native template DNA. It does this by utilizing dUTP instead of dTTP during the PCR reaction, thus the nucleotide bases in the PCR products will contain uracil instead of thymidine. Then, before starting your subsequent PCR reaction, samples are pretreated with UNG, which hydrolyses uracil-glycosidic bonds in DNA amplicons containing dUTP, thereby degrading the contaminant PCR products into small fragments, while having no effect on free dUTP or native template DNA containing thymidine.
UNG is then heat denatured and inactivated prior to initiation of your qPCR reaction. New PCR products will contain dUTP and thus be amenable to carry-over prevention on subsequent reactions.
Master mix optimization: The dyes
When performing non-probe based qPCR, an essential component of your qPCR master mix is the use of dyes. This may include the use of a passive internal reference dye (such as ROX) for normalization of background fluorescence in instruments that can be susceptible to well-to-well variations.
Additionally, when not using sequence specific DNA probes, your master mix may include a non-specific double stranded DNA binding dye, such as SYBRⓇ Green. These particular dyes can be ideal for screening assays, single target identification including genotyping, or when running smaller numbers of assays. Additionally, there are non-specific double stranded DNA binding dyes, such as the Roche ResoLight Dye, that bind to DNA in a saturated manner without inhibiting the PCR reaction like other non-specific dyes. This results in highly homogenous labeling of amplicons in real-time during DNA amplification, and are therefore amenable to high-resolution melting (HRM) analyses.
Carefully evaluating your master mix ingredients is essential to know whether you are getting the most from your reagents and maximizing the potential for success of your assays. So whether you are performing small- or large- scale qPCR assays for genotyping, or gene expression quantification, there is a master mix available to help you reach your experimental goals.
1. Brock TD, Freeze H. Thermus aquaticus, a Nonsporulating Extreme Thermophile. J. Bact (1969). 98 (1): 289–97.