Fluorescent Probes – The Shining Star of PCR

Application and Synthesis of fluorescent Probes

 

What are DNA probes?


The term „probes“, is commonly used to describe fluorescent oligonucleotides. The most used probes are dual-labeled and thus consist of three distinct parts:

a) a single strand DNA between 5 and 35 bp responsible for specific DNA recognition
b) a reporter dye emitting fluorescence at a certain wavelength
c) a quencher absorbing the light emitted by the reporter dye at a certain state.

DNA probes are labelled with an appropriate pairing of reporter dye and quencher, to utilize FRET, (Förster resonance energy transfer) a well-known quenching mechanism depending on the distance of emitting dye and absorbing quencher.

As long as probes are floating in solution, they can freely form coils or hairpins, in which the quencher stays close enough to the reporter dye quenching any emission. However, as soon as probes bind on their target DNA, reporter dye and quencher get separate.

Depending on the length of the probes already the separation through binding is sufficient to neutralize the quenching effect allowing light emission. Probes that emit after binding are called hybridization probes. The best known are Molecular Beacon, Scorpion, Amplifluor® and LUXTM, that all form hairpins to stabilize the quenched state (see Figure 1).

Figure 1: Principle of Molecular Beacon Probe. During hairpin formation (left) the quencher (blue) stays very close to reporter dye (green) and thus fluorescence emission is inhibited. Binding to the amplified target DNA (right) change the probe conformation separating reporter dye and quencher to allow fluorescence emission detecting the target DNA.

In case of short DNA probes, the separation of reporter dye and quencher occurs only after a polymerase hydrolyzes the probe releasing the reporter dye. Probes that need to be hydrolyzed are named TaqMan Probe based on the TaqMan polymerase with
3´-5´exonuclease activity (see Figure 2).

Figure 2: Principle of TaqMan Probe. In case of short dual-labeled oligos (left), the distance of reporter dye (green) and quencher (blue) is sufficient small to inhibit fluorescence emission, even after binding to the amplified target DNA. During the elongation phase of the PCR, Taq DNA polymerase with exonuclease activity cleaves the reporter dye from the oligo, allowing fluorescence emission detecting the target DNA.

Why are fluorescent probes used?


One of the major uses of probes is to visualize targeted DNA sequences for quantitative real-time PCR (qPCR), which became a exremely versatile tool due to the probes.

Quantitative PCR can also be performed with fluorescence dyes binding to dsDNA, whose amount increases during the PCR. However, these dyes bind to any dsDNA without distinguishing target DNA from other DNA strands. In contrary, probes show higher specificity due to specific oligo part annealing only on the target sequence.

The hybridization probes allow further applications, beside quantification of pathogens also mutation detection, melting point analysis and in situ hybridization. Furthermore, probes can be labeled with different reporter dyes and thus allow multiplexing: detecting multiple targets in a single reaction.

 

How are fluorescent probes synthesized?


The most popular probes are TaqMan probes and Molecular Beacon, both dual-labelled with a 5´-reporter dye and a 3´-quencher for FRET.

The first dual-labeled probes available for PCR were synthesized in two steps. At first a target specific oligo was synthesized with a 3´-amino group and a 5′-reporter dye using the common solid phase chemistry with phosphoramidites whereby the last nucleotide was further modified with the reporter dye. After releasing the oligo from the solid phase, the quencher was added by coupling an active ester to the 3′-amino group. As the post-synthetic coupling of the quencher was not fully efficient, the obtained probes needed to be purified not only from incomplete oligos but also from probes, which are not quenched. Both the post-synthetic modification and the purification limited the evolving of probes for PCR initially.

Then controlled pore glass (CPG) modified with 3′-quencher got available as synthesis support columns allowing solid state synthesis of oligos that start directly with a nucleotide 3′-quencher. The outcome of the solid-phase synthesis is directly a dual-labeled oligo requiring the same purification steps as common primers in terms of extraction of full-length probes. Thus, the introduction of CPGs with 3′-quencher simplified the synthesis of probes significantly making it affordable to any DNA synthesis laboratory.


Improvements, types and costs of usual probes


Further improvement of the synthesis provided the introduction of the first non-fluorescent quencher, Black Hole Quencher 1 (BHQ-1TM) by Biosearch Technologies. Beside the lack of intrinsic background fluorescence, the BHQ-1 quencher requires, in contrast to other quenchers such as TAMRA, no changes to standard oligonucleotide synthesis procedures. Therefore, it was possible to complete the synthesis and deprotection of a dual-labeled FRET probe on a standard automated DNA synthesizer.

Today a plenty of numbers of quenchers are available. The most outstanding is the BlackBerry® Quencher (BBQ) that can be combined with all fluorescent dyes allowing the synthesis of different fluorescent probes with the same CPG.

The special reagents and materials as well as the high demands on the probe quality for sensitive qPCR contribute to the high costs of probes, typical around €150/180$ to €250/300$ per target.

The costs increase even more for DNA probes with more complex features than the common dual-labeled FRET probes as they still require proper post-synthetic modifications.

Why Kilobaser moves your research forward – especially now, with our new probe synthesis?


Using our know-how in DNA chemistry in combination of our device for microfluidic chip technology, we take on the challenge to synthesize the fluorescent probes for qPCRs.

The biggest advantages of our Kilobaser benchtop DNA synthesizer are:

  • speed
  • user-friendliness
  • low consumption of reagents
  • high efficiency of the oligo synthesis
  • data privacy

Kilobaser incorporates a microfluidic and a novel reagent cartridge technology and can produce ready-to-use DNA primers in less than 2 hours. This technology produces 50% less hazardous waste than usual column-based synthesizers. The typical output of a 20mer primer is a DNA sequence ≥ 85% full length product without any purification process. Besides that, Kilobaser ensures complete Data Privacy and a maximum of user-friendliness.

These advantages now also apply for Kilobaser probe synthesis, except 2 differences:

  •  Kilobaser DNA probes 100 times faster than ordering online: Instead of waiting for 4-8 working days for probes from online synthesis provider, have your desired DNA probes ready in less than 2 hours with Kilobaser.
  • Cheaper: Kilobaser DNA probes are not just a lot faster but also cheaper than ordering from online synthesis provider.

Request a product demo and ask us for a cost evaluation for your individual situation.

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