Probe Systems For Real-Time PCR

Most real-time PCR assays begin with some sort of fluorescent reporter molecule(s). The simplest example of this is an intercalating dye from Molecular Probes called SYBR® Green. Specific for double-stranded DNA (dsDNA), SYBR® Green emits light upon binding DNA, and thus can be used to quantify the amount of PCR product. SYBR® Green is easy to use, cheap, and sensitive. But the disadvantage of SYBR® Green is that it binds to any dsDNA, including non-specific reaction products. In addition to potentially causing problems with quantification, the fact that SYBR® Green binds to any dsDNA makes it inappropriate for genotyping applications where single nucleotide differences must be detected.

For these reasons, many people choose to use hybridization probe systems based on fluorescence resonance energy transfer (FRET). Unlike SYBR® Green, FRET-based probe systems are well suited to a range of applications. “Hybridization probes allow the discrimination between closely related sequences on the basis of a melting curve analysis carried out subsequent to the PCR reaction,” says Tobias Ruckes, associate marketing director of molecular diagnostics for Qiagen. “This technology is useful for diagnostic purposes such as genotyping (SNP analysis) or virus subtyping.”

There are different kinds of FRET-based probe systems, each of which has its own advantages. TaqMan® and molecular beacons are two of the most popular systems. “TaqMan® and molecular beacon formats are particularly good when specificity is needed,” comments Mark Behlke, vice president of molecular genetics and biophysics at Integrated DNA Technologies. “This includes applications interrogating SNPs or when performing quantitative expression assays on targets that may be members of multi-member gene families or have expressed processed pseudogenes. The high specificity of these assays makes them great choices for all applications.”

Tania Nolan, business development manager in molecular biology at Sigma-Genosys, also differentiates the various probes according to application. She recommends using TaqMan® for general quantification, and TaqMan® LNA (see below) as an “excellent choice for SNP detection, [because it] increases sensitivity in some cases, when a short probe is required [such as for] pathogen detection.” She favors molecular beacons for in vivo studies and genotyping, and Scorpion™ probes (see below) for SNP analysis and haplotyping. Nolan says that Scorpion™ probes confer very sensitive detection, as “when the sequence is a minority sequence in a background of a similar majority sequence, [such as with] mutation detections.”

TaqMan® probes are not appropriate for all applications, though, as they “do not allow for reliable melting curve analysis, which renders them less suitable for genotyping purposes,” says Ruckes. And because conventional TaqMan® probes are relatively long, “single nucleotide exchanges (as in SNP analysis) would not reliably be detected.” However, a derivative of TaqMan® probes, called minor groove binder (or MGB) probes, have “been designed for exactly this purpose,” says Ruckes. “MGB probes are much shorter in length (12-15 nt) with chemical compounds attached to their 3' ends, which bind to the minor groove of the DNA. The binding of this MGB portion compensates for the lower melting temperature of the probe. The short probes allow sequence discrimination on basis of single nucleotide exchanges.”

Applied Biosystems offers their popular Custom TaqMan® TAMRA probes, dual-labeled with the 5’ reporter dye of your choice (Fam, Vic, or TET dye) and the 3’ TAMRA quencher dye. This probe is most often used for real-time applications such as quantitative gene expression and pathogen detection. Applied Biosystems’ Custom TaqMan® MGB Probes are also dual-labeled, using the 5’ reporter dye of your choice (Fam, Vic, TET, or NED dye) and a 3’ non-fluorescent quencher, which results in relatively lower background signal. The MGB moiety gives greater stability to the hybridized probe, which raises its melting temperature during amplification. As a result, MGB probes can be effective at lengths shorter than traditional dual-labeled probes, making them better suited to applications such as SNP genotyping and other allelic discrimination applications. Regina Nagle, product manager of gene expression assays for Applied Biosystems, says that “Applied Biosystems’ DNA sequencers are based on 4 FRET dyes to represent 4 bases. Currently, Applied Biosystems’ sequence detection systems are mainly based on TaqMan® probes.”

Sigma-Genosys also offers an improvement on the old TaqMan® workhorse—LNA Dual-Labeled Fluorogenic Probes. LNA (locked nucleic acid) is a type of nucleic acid analog that confers greater thermal stability to the probe, reducing background fluorescence and increasing the duplex melting temperature (as for the MGB probes, but by a different mechanism). The end result is that the probes can be smaller, which is more effective in studying single nucleotide changes (such as with SNP genotyping analysis) or in otherwise problematic target sequences. These probes are available with the 5’ reporter dyes 6-FAM, HEX, or TET, and the 3’ quencher molecules TAMRA, DABCYL, BHQ-1, or BHQ-2.

Another supplier of molecular beacons, Integrated DNA Technologies (IDT), strives to offer superior quality control. All IDT probes undergo quality control by at least one type of mass spectrometry, says Behlke: “No other manufacturer offers this combination of QC, or on such a wide scale.” Another attractive feature about IDT’s probes is their wide range of fluorophores and dyes, and their ability to make quality custom products to your specifications. They have recently developed a new series of dark quenchers called Iowa Black FQ and Iowa Black RQ. “The FQ modification will quench fluorophores in the Fam–Cy3 spectral range, while the RQ modification will quench fluorophores in the Tamra–Cy5.5 spectral range,” explains Behlke. “These quenchers have strong hydrophobic character which provides a convenient ‘handle’ to assist in rapid purification using RP-HPLC. Dark quenchers of this kind provide lower background and also enable multiplex reactions using different reporter fluorophores.”