Probe qPCR detection chemistry involves the use of an oligonucleotide probe that contains attached fluorophores to allow for fluorescence detection of a specific amplified target sequence. Within this group exists three major classifications: primer-probes, hydrolysis probes and hybridization probes. The structures, along with the advantages and disadvantages of each, vary by type. Understanding these differences will allow you to choose the best probe for your qPCR assay.
Scorpions
Scorpion probes are classified as primer-probes, since they contain both a primer and a probe within the same molecule. Within this primer-probe classification, they are more specifically described as hairpin primer-probes.
When Should You Choose Scorpions?
Due to the unique unimolecular structure of Scorpions, these primer-probe molecules provide extremely fast detection of PCR products. Their structure also aids them in producing an excellent signal-to-noise ratio. These characteristics make Scorpions great options if your assay priorities are shorter reaction times, strong signals and enhanced discrimination. Additionally, Scorpions are a good choice when cost is an important consideration, as they don’t require purchasing both a primer and a probe.
- Unimolecular → Fast reaction kinetics
- No primer-dimer formation
- High specificity
- Strong signals & minimal background noise
- Multiplexing
Structure
This hairpin structure contains a reporter fluorophore at the 5’ end of the single-stranded oligonucleotide and a quencher probe at the 3’ end of the oligonucleotide. The loop portion of the hairpin contains a base sequence that is complementary to the sequence of the target DNA.
Additionally, a primer is attached to the hairpin structure. A hexathylene glycol (HEG) blocker attaches the 3’ end of the hairpin to the 5’ end of the primer. The HEG blocker ensures the primer won’t be copied by the polymerase.
Function
In this closed loop conformation, the intensity of fluorescence emitted by the reporter fluorophore is greatly reduced due to its quenching by the nearby quencher fluorophore, a process known as fluorescence resonance energy transfer (FRET). Following denaturation, the probe binds to the complementary sequence of the new DNA. When the probe binds to its complementary region of an amplicon, the hairpin structure opens and the reporter fluorophore separates from the quencher fluorophore. This separation of the reporter fluorophore from the quencher fluorophore allows the detection of the fluorescence signal from the reporter fluorophore to intensify. This increase in intensity occurs in proportion to the increase in the amount of amplicon.
Other primer-probes: Cyclicon, LUX
TaqMan
TaqMan probes are classified as hydrolysis probes. These probes are also classified as bimolecular, as the primers and probes are not attached within a single molecule.
When Should You Choose TaqMan?
TaqMan and other hydrolysis probes are normally your best option when cost is your top consideration, as they tend to be more cost-friendly. They are also tremendously dependable, making them a good option if you’re looking to perform high-volume assays.
- High specificity
- Multiplexing
- Easy probe design
Structure
TaqMan probes consist of a single-stranded oligonucleotide that is synthesized to bind to a specific single-stranded target DNA sequence. They contain a reporter fluorophore attached at the 5’ end and a quencher fluorophore attached at the 3’ end.
Function
The TaqMan probe anneals to the target region specified downstream of the primers used in the assay. As the DNA polymerase extends the primer and continues to synthesize the new strand, the reporter fluorophore of the probe becomes separated from the quencher fluorophore via cleavage of 5’ to 3’ Taq DNA polymerase exonuclease activity. The intensity of the fluorescence detected from the reporter dye is no longer quenched by the nearby presence of the quencher fluorophore via FRET. The probe is removed from the target strand as the polymerase continues to extend the strand. As the amount of amplified product increases and additional probes are cleaved, the fluorescent signal measured will increase proportionally.
Molecular Beacon
Molecular Beacon probes are a type of hybridization probe. They are single-stranded oligonucleotides containing a hairpin loop structure.
When Should You Choose Molecular Beacon?
If one of your top assay priorities is minimizing background noise and very high specificity, Molecular Beacon probes are an excellent option. In situations where your target sequence is highly uncommon, this can be very important.
- Very high specificity
- Multiplexing
- Probe conservation
Structure
The structure of Molecular Beacon probes consists of four distinct regions. The loop portion contains the base pairs that are complementary to the target DNA sequence. The stem region of the hairpin contains two sequences, one at the 5’ terminus of the probe and one at the 3’ terminus of the probe, which are complementary to one another. Both ends of the stem region are attached to the loop region. The probes also contain a reporter fluorophore at the 5’ end and a non-fluorescent quencher fluorophore at the 3’ end.
Function
When the hairpin is in its closed conformation, the fluorescence emitted by the reporter fluorophore is quenched by the quencher fluorophore. Following denaturation of the target DNA and annealing of the primers, the hairpin probe unfolds and binds to the target sequence. The fluorescent emission from the reporter probe is no longer quenched, because the quencher fluorophore is no longer in such close proximity to the reporter probe. The fluorescent signal emitted by the reporter fluorophore is detected, and increases proportionally with the increase in PCR products produced. Since the hairpin loop will only unfold in response to an exact complementary sequence, these probes may be used when very high degrees of specificity are required.
Other hybridization probes: FRET, Eclipse