by Caitlin Smith
Real-time PCR, or quantitative PCR (qPCR), quickly became an important evaluative tool for researchers needing to quantify the product of a PCR reaction—yet it is an unfinished technique. Despite its widespread use, or perhaps because of it, qPCR is still challenged to grow. “Real-Time PCR is widely regarded as the technology researchers use to validate novel genomic discoveries and is currently being used to study most types of genomic events, including long and small RNA expression, copy number variation, and methylation,” says Allen Nguyen, product manager for TaqMan® assays at Life Technologies. “New applications are still emerging for employing real-time quantitative PCR (qPCR) to complement upstream de novo discoveries. Analysis of non-coding RNAs is a great example of this type of new and exciting application area. Whereas non-coding RNAs were previously considered ‘junk’ nucleic acid, researchers now know that they are heavily involved in cell regulatory processes and disease mechanisms.”
Many find they are using qPCR more and more—especially as technology is smoothing out some reagent and methodological wrinkles. For example, one of the main challenges in qPCR is quantifying the reaction product. Two common methods for this include the use of nucleic acid probes, or the use of dyes to signal the amount of reaction product. This article focuses on some recent developments in probes and dyes that are used to indicate the amount of final product present in a qPCR reaction mixture.
Using probes for quantification
The goal of your particular experiment can help you weigh the pluses and minuses of probes versus intercalating dyes, as both can have distinct advantages. For example, probes detect only specific amplification products. They bind to DNA and their fluorescent signal is detectable when the DNA polymerase moves by and cleaves off the probe’s quencher molecule. Because probes are specific for their targets, this method can reduce background and false positives. On the other hand, probe specificity means you must make (or order) a different probe for each target sequence, costing time and money.
Life Technologies supports both types of qPCR detection, but its latest offerings are based on its TaqMan® probe technology. Their new Applied Biosystems TaqMan® Non-coding RNA Assays and Applied Biosystems TaqMan® Pri-miRNA Assays help researchers to study specifically the long non-coding RNAs and primary microRNA transcripts. The assays “were designed using the same algorithms as our industry-leading TaqMan® Gene Expression Assays, but the algorithms were tuned to only allow very specific detection of the non-coding target transcript and not any coding RNAs,” says Nguyen. “In the new paradigm of prolific RNA expression with lots of complex cellular networks of overlapping coding and non-coding RNAs, researchers need ultra-specific tools to unravel and characterize the relationships between coding and non-coding transcripts. It takes a lot of bioinformatics knowledge and real-time PCR assay design expertise to ensure that the results that researchers obtain are specific and accurate, which comes from robust assay designs.”
Nguyen explains that it can be challenging for researchers to understand “how and when to use the different real-time PCR technologies available. There are several choices for real-time PCR vendors and the chemistries they offer, including the probe-based platforms like TaqMan® and DNA intercalating dye-based like SYBR Green.” He adds, “Researchers who need the added layer of specificity from a probe and don't want to compromise their results to reduce costs or spend time designing their own assays will tend to go with TaqMan® assays.”
Using SYBR Green for quantification
In contrast to specific probes that must be synthesized for each target, dyes as detection tools can be easier to use. Dyes such as SYBR Green I, that intercalate into double-stranded DNA (or for other dyes, bind to the DNA minor groove) are more convenient to have on hand, and less expensive, than probes. The disadvantages of dyes include detection of non-specific double-stranded reaction products (resulting in increased background or false positives). In addition, some dyes are known to inhibit the PCR reaction.
Nonetheless, many companies offer kits or master mixes containing the widely used dye SYBR Green I as a detector of amplification product in qPCR reactions. For example, Roche Applied Science’s LightCycler® 480 SYBR Green I Master is a hot start reaction mix for qPCR that includes their FastStart Taq DNA Polymerase, as well as SYBR Green I dye, for use in their LightCycler® 480 Instrument. Qiagen also includes SYBR Green in their QuantiFast and Rotor-Gene SYBR Green PCR and RT-PCR kits.
SYBR Green I alternatives
For those who want the convenience of using a dye as a qPCR detector, other dyes are available besides SYBR Green I that offer its benefits along with fewer of its disadvantages. Paul Monis, adjunct senior lecturer in the School of Pharmacy and Medical Sciences at the University of South Australia, uses a dye called SYTO9 for qPCR. “I first started using it back in 2000. It is better than SYBR Green,” he says. “It doesn't cause PCR inhibition, [and it’s] better for DNA melting curve analysis. We have evaluated some of the newer dyes, but none have been that much better than SYTO9 that we've changed.” In 2005, Monis et al.1 published a comparison of SYBR Green I and SYTO9, finding that the latter is less inhibitory to PCR, and gives highly reproducible DNA melting curves; yet SYBR Green I is still the dominant dye used in kits sold for qPCR. “The biggest challenge is getting people to change,” adds Monis. “There is no logical reason to persist with SYBR Green—it is inferior to every other dye now on the market.” Similarly, another research group from Denmark2 found that SYBR Green alternatives SYTO-82 and SYTO-13 do not inhibit PCR, show preferential binding to GC-rich regions, or influence melting temperature (as did SYBR Green I in their hands).
While also offering SYBR Green tools for qPCR, Bio-Rad now has a SYBR alternative dye called EvaGreen. According to Viresh Patel, senior product manager for reagents at Bio-Rad, “SYBR Green alternatives have contributed to improvement in qPCR performance, while also expanding the application areas for these types of reagents.” Bio-Rad combines EvaGreen with their Sso7d fusion polymerase and optimized buffer for better qPCR performance, into their SsoFast EvaGreen Supermix. “EvaGreen is a third generation dsDNA binding dye which offers several advantages over SYBR Green,” says Patel. “It is less inhibitory to PCR and can be used under saturating conditions to generate greater fluorescent signals. EvaGreen is also well suited for HRM [high resolution melt] analysis.”
Singleplex qPCR continues to challenge scientists to “maintain sensitivity, specificity, and reproducibility under challenging conditions,” adds Patel. “This can include increased throughput and reducing run times (fast PCR), lower costs and lower reaction volumes, or bypassed nucleic acid sample preparation (direct PCR). High quality reagents, enzymes, and instruments are available. However, further advancements in both hardware and reagents are required to overcome these challenges.”
References
1Monis PT, et al. "Comparison of SYTO9 and SYBR Green I for real-time polymerase chain reaction and investigation of the effect of dye concentration on amplification and DNA melting curve analysis." Anal Biochem, 340: 24 – 34, 2005.
2Gudnason H et al. "Comparison of multiple DNA dyes for real-time PCR: effects of dye concentration and sequence composition on DNA amplification and melting temperature. Nucleic Acids Res. 35: e127, 2007.