qPCR Probes -
For Probe-Based qPCR Assays

The classical Dual Labeled Probes are hydrolysis probes that are designed to increase the specificity of quantitative PCR.

Dual Labeled Probes used in quantitative real-time PCR systems take advantage of the 5' -> 3' exonuclease activity of Taq polymerase. They consist of a fluorophore at the 5'-end and a 3' quencher which anneals between the PCR primers. 

During the extension phase of PCR, the 5' -> 3' exonuclease activity of Taq polymerase cleaves the fluorescent reporter from the probe. The amount of free reporter accumulates as the number of PCR cycles increases. The fluorescent signal from the free reporter is measured in real-time and allows the quantification of the target sequence.

Available dye-quencher combinations:

Product specifications:

• Available synthesis scales: 0.01 - 1.0 µmol
• Probe lengths: 10 - 40 bases (wobbles non-defined ratio possible)
• HPLC purified by default
• TAT: 3 – 5 working days (1.0 µmol scale takes longer)

MGB Probes provided by Eurofins Genomics stabilise probe-target hybridisation and increase melting temperature.

Eurofins Genomics MGB Probes incorporate a 5’ fluorescent dye and the 3’ Eclipse quencher (EQ) attached to the minor groove binder (MGB) molecule.

Probes containing a MGB molecule form an extremely stable duplex which gives the probe a significant increase in the melting temperature (T_m). It is therefore possible to use shorter probes (down to 13 bases) in real-time PCR compared to conventional dual-labeled probes.

MGB Probes for gene expression analysis & SNP genotyping

The double-stranded DNA is denatured by raising the temperature. At this point the fluorescent dye of the probe is quenched by the MGB-Eclipse quencher.

Figure 1: Gene Expression analysis (click to enlarge)

By decreasing the temperature the PCR primers and MGB probe anneal to their specific target sequence. Allelic discrimination is achieved by the selective annealing of the MGB probes (Figure 2).

The taq polymerase synthesises a complementary DNA strand using the PCR primers and template as a guide. When the polymerase reaches the MGB probe its  5´nuclease activity cleaves the probe and separates the fluorescent dye from the non-fluorescent quencher.

Figure 2: Allelic discrimination by selective annealing (click to enlarge)

Available dye-quencher combinations:

Product specifications:

• Selectable delivered quantities of 5 nmol, 20 nmol and 40 nmol
• Probe lengths: 10 - 40 bases
• HPLC purified by default
• TAT: 3 – 5 working days

LocNA Probes facilitate the optimal design of highly specific, shorter probes.

LocNA probes contain Locked Nucleic Acid (LNA) bases which improve the stability and performance of qPCR assays. These modified bases provide a 2'-O, 4'-C methylene bridge which effectively "locks" the 2' oxygen to the 4' carbon. LocNA probes are the optimal choice when designing highly specific, shorter probes with challenging target sequences.

Available dye-quencher combinations:

Product specifications:

• Average synthesis yields of 15 nmol resp. 2 OD
• Sequence length from 10 - 40 bases
• Up to 15 LocNA bases per probe are possible
• Only DNA bases are allowed at the 5' and 3' end
• HPLC purified by default
• Delivered lyophilized or concentration adjusted in tubes
• Production takes 5 working days

Molecular Beacons are hairpin-shaped hybridisation probes.

The sequence of these probes is designed to allow the formation of a hairpin structure in which the fluorescent dye and the quencher are in close proximity. The probe is designed to anneal between the PCR primers.

When the probe hybridises to its target sequence in the PCR annealing step, the loop opens and the fluorescent reporter and quencher are separated, resulting in a fluorescent signal upon excitation. The amount of signal is proportional to the amount of target sequence, and is measured in real time to allow quantification of the amount of target sequence.

Molecular Beacons can be ordered as Dual Labeled Probes.

LightCycler probes or FRET probes are hybridization probes based on the fluorescent resonance energy transfer (FRET) technique.

They consist of a pair of oligonucleotides (donor and acceptor) each labeled with a different fluorescent dye.

Donor and acceptor are designed to hybridise to adjacent regions in close proximity to each other on the target DNA. The acceptor probe is labeled with a fluorescent dye at the 5' end and the donor probe with fluoresceine at the 3' end. Interaction of the two dyes can only occur when both dyes are bound to their target.

During FRET, the fluorescent donor molecule is excited by an external light source which subsequently transfers its energy to the acceptor fluorophore. The excited acceptor emits light of a different (longer) wavelength, which can be detected and measured.

Acceptor

Donor

Please order these probes as Custom DNA Oligos >> here!

BHQ1 [BHQ1] 480-580 nm

BHQ1 is one of the Black Hole Quenchers from Biosearch Technologies, Inc. BHQ1 is a highly efficient dark quencher for Dual Labeled Probes, Molecular Beacons and FRET probes with an excellent spectral overlap for all dyes in the green to dark yellow emission spectrum.

BHQ2 [BHQ2] 520-650 nm

BHQ2 is the second Black Hole Quencher from Biosearch Technologies, Inc. in our portfolio. BHQ2 is a highly efficient dark quencher for Dual Labeled Probes, Molecular Beacons and FRET probes containing 5’ fluorophores in the dark green to orange emission sector.

BBQ 650 [BBQ650] 550-750 nm

The BlackBerry Quencher 650 is an excellent long-wavelength quencher for Dual Labeled Probes, Molecular Beacons and FRET probes containing long wavelength dyes such as Cy5, Cy5.5 and other dyes in the red to near-infrared emission spectrum.