Real-time quantitative PCR has undoubtedly hit its stride as the go-to protocol for assessing gene expression. When it emerged about 10 years ago, this gene-quantifying technology introduced vast improvements over its time-consuming, multi-step frontrunner, end-point quantitative PCR. Sophisticated fluorescent chemistry—as well as technologies to detect these light-emitting molecules—simplified the process, and, thus, opened the door to its ubiquity in research labs and increasing presence in clinical labs.
“It’s a pretty good technique for what customers want to use it for: gene expression analysis,” says Jeremy Gillespie, PhD, group product manager at ThermoFisher, which has a growing line of real-time PCR reagents.
Improvements in reagents have a lot to do with the protocol’s spread and current popularity. The original ethidium bromide—an intercalating dye that non-specifically binds to double-stranded DNA—has been left behind. A number of newer fluorescent probes were quickly developed, and gained fans for offering remarkable levels of sensitivity, reliability, and dynamic range. These probes are designed to emit light only after binding to target gene sequences.
Among the earliest and most popular of these probes are Life Technologies/Applied Biosystems’ TaqMan® probes. Labeled with both a fluorescent reporter and a quencher, TaqMan probes fluoresce when Taq DNA polymerase and its 5’ nuclease activity cleaves apart the probe. Separation of the quencher allows release of fluorescent signal from the reporter. Roche Applied Science’s HybProbes depend on a pair of probes, one with a donor fluorophore and another with an acceptor fluorophore. Designed to anneal in close proximity on the target, light emitted from the donor causes the acceptor fluorophore to emit light, which is detected and recorded.
Another option is the hairpin-shaped probe. One example is Life Technologies/Invitrogen ’s Light Upon eXtension technology. These primers take on a hairpin structure such that the fluorescent reporter at one end is held in close proximity to the quencher at the opposite end. Upon binding to the target, the hairpin unfolds, allowing release of fluorescence signal. These primers are known for their ability to detect less than 100 copies of target genes.
One of the main advantages of using sequence-specific probes is the ability to multiplex, or simultaneously assess the expression of multiple genes. It’s a capability that’s ever more important in today’s climate of high-throughput strategies. By using fluorophores of different colors and/or emission spectra for each target, you can compare the expression levels of various genes all under the same reaction conditions.
However, the advantages of specificity come at a cost, says Stephen Gunstream, who directs the real-time PCR division at Integrated DNA Technologies. “There’s a huge difference in price between intercalating dyes and the higher quality assays, like the 5’ nuclease assay,” he says, referring to the hydrolytic action that releases the fluorescence.
Thus, SYBR® Green, the most common intercalating dye in today’s labs, maintains a broad customer base. Its non-specific binding nature means that SYBR Green molecules not only bind to target genes that have been amplified, but also to primer-dimer artifacts and the products of amplification errors. Despite this and other problems, the advantages of SYBR Green seem to weigh significantly in its favor. In addition to the low cost, researchers enjoy the relative ease of use and the freedom from having to design sequence-specific probes.
“The design of sequence specific probes is not as straightforward, and cost is an important consideration,” says Scott O’Brien, product manager at Stratagene, a division of Agilent Technologies. “Using SYBR Green is a simple universal solution for all gene targets.”
Stratagene recently released its second generation of master mixes dubbed Brilliant II. These reagents are available in SYBR Green and probe format with newer mixes formulated specifically for fast quantitative PCR, one of the fastest growing areas for real-time PCR.
In answer to budget-conscious researchers, Integrated DNA Technologies has recently introduced PrimeTime Mini in an effort to lower the cost of using sequence-specific probes. Instead of paying $120 for the company’s standard real time PCR reaction mix of 500 reactions, the mini version provides enough primers and probes for 100 reactions at $75.
The new product, in part, caters to researchers who need to validate findings discovered during high-throughput screening, Gunstream says.
“Customers only want to do about 50 reactions to validate hits from microarrays,” he says. “We make it much more affordable to do that.”
Roche is also trying to lower the obstacle of cost for researchers. Their Universal ProbeLibrary circumvents the need for expensive, custom-made probes. Instead, the company offers 165 probes that can bind to the most prevalent transcripts of the transcriptomes of 11 commonly studied species. Like TaqMan probes, these are hydrolysis probes. The difference is that they are made up of eight to nine nucleic acids each, some of which are locked nucleic acids, which are DNA nucleotide analogues that bind with greater strength than standard nucleotides.
“It’s a really clever, elegant, yet simple solution,” says Mike Leous, marketing manager for Roche Applied Science. “And the free online software designs assays in minutes, eliminating any perceived ease-of-use advantages for SYBR Green.”
From probes and probe libraries to SYBR Green, real-time PCR reagents continue to keep pace with today’s, and tomorrow’s, research advances.