DNA Probes

Overview

DNA Probes in a nutshell

In simple terms, DNA probes are specifically chosen, short nucleotide sequences with a reporting modification on at least one end. For the majority of applications, DNA probes are dual labeled with reporter and quencher modification.

The 3 building blocks

Single Stranded DNA; 15-35 bp long; specific to target DNA

Reporter dye; emitting a certain wavelength

Quencher; absorbing the emitted wavelength

The Probe Principle

DNA probe Reporter not emitting in near distance to DNA probe Quencher.
DNA probe Reporter emitting in far distance to DNA probe Quencher.

Like a bad neighbor, the quencher absorbs all signal emitted by the reporter. For this to happen, reporter and quencher have to be very close to each other.

Once reporter and quencher move apart, the signal is no longer absorbed and can be detected.

Therefore, the connecting DNA strand must be constructed in such a way that the distance between reporter and quencher can change during a reaction.

The variety of designs results in the different types and applications of DNA probes.

Probe Application

TaqMan probe binding to target DNA. Polymerase hydrolizing reporter to seperate it from DNA strand. Reporter signal is no longer absorbed and can now be detected.
Molecular Beacon probe binding to target DNA. This seperates Reporter an quencher to a distance, so the emitted reporter signal is no longer absorbed from quencher.

DNA probes, for example TaqMan and Molecular Beacon probes, are mostly used for quantification and detection applications.

possible methods & Applications:

RT-qPCR

Real-Time/qPCR

Multiplex PCR

Pathogen detection

Gene detection

Gene mutation detection

Gene localisation

The Synthesis – Step by step

Single stranded DNA.

First, it is important to understand that a DNA strand has two ends, which differ in their biochemical properties. These are called the 3′-end and the 5′-end.

Quencher bound on CPG column during DNA probe synthesis. Nucleotide bases in solution about to bind on Quencher.

Thanks to Controlled Pore Glass (CPG), we can anchor molecules of our choice directly to the solid phase, i.e. the column. As a result, each DNA synthesis begins with a modification of one’ s choice.

DNA probe, with quencher, bound on CPG column during synthesis. Nucleotide base in solution about to bind on DNA sequence.

Since DNA synthesis runs from 3′-end to 5′-end, the solid phase anchored modification, the quencher, is located on the 3′-end of the DNA probe. The DNA probe grows with each nucleotide base added to the DNA strand.

DNA probe, with quencher, bound on CPG column during synthesis. Reporter in solution about to bind to DNA sequence.

Once the sequence is fully synthesized, it is time to bind the second modification, the reporter, at the 5′-end.

DNA probe, with quencher and reporter, bound on CPG column during synthesis.

Finally, the basic structure of the DNA probe is completed. Just a few more chemical steps and we get a ready-to-use DNA probe!

In order to get more in-depth understanding, you can find more on our blog titled “Fluorescent Probes – The Shining Star of  PCR

DNA Probe Synthesis with Kilobaser

Ready-to-use DNA probes in 3 steps

enter sequence

Enter your DNA sequence using the included touch screen, USB flash drive or your computer.

insert chip & cartridge

Insert Kilobasers' innovative microfluidic chip & reagent cartridge for DNA probe synthesis.

Press start

Within two hours you get your ready-to-use DNA probe!

Discover more about how Kilobaser helps smoothen your lab routine!

FAQ – DNA Probes

What do I need to labele my probe with 6-FAM Reporter?

With our 6-FAM reagent cartridge you are able to synthesize DNA probes automatically labeled on 5'-end with 6-FAM Reporter. You will not need any extra reagents.

How can I add BHQ-1 to my probe?

Use our BHQ-1 Microfluidic chip to synthesize DNA probes labeled on 3'-end with BHQ-1 Quencher. You do not need to add extra reagents, since BHQ-1 is already anchored to the column in the chip.

Can I synthesize Molecular Beacon probes with Kilobaser?

Yes, you absolutely can! As reagent cartridge, use the 6-FAM cartridge and combine it with the BHQ-1 microfluidic chip. As a result, your DNA probe will be labeled with a 6-FAM reporter at 5'-end and with a BHQ-1 quencher on 3'-end.

Can Kilobaser synthesize TaqMan probes?

Yes, it can! Use the 6-FAM reagent cartridge in combination with the BHQ-1 microfluidic chip. With this combination, your DNA probe will be labeled with a 6-FAM reporter at 5'-end and with a BHQ-1 quencher on 3'-end. 

TaqMan probe binding to target DNA. Polymerase hydrolizing reporter to seperate it from DNA strand. Reporter signal is no longer absorbed and can now be detected.

You have further questions? Maybe you will find the answer in our FAQ-section.

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Lisa-Marie Michelitsch​

Master of Science
Biotechnology

sales@kilobaser.com
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