PCR probes are DNA or RNA sequences labeled with a reporter molecule. The reporter is a molecule that emits fluorescence when hybridized to the target sequence.
These probes are used in quantitative and real-time PCR methods such as qPCR or quantitative PCR, which allows real-time amplification detection during the PCR process.
Several PCR probes are available commercially, so how do you choose a PCR probe for your experiment? We’ll explore this and other questions in this article.
In this article:
Difference between a PCR probe and a primer
If you want to study a target sequence, how do you differentiate it from the other sequences in a pool of DNA or RNA? This is what the PCR probe was designed to help with.
PCR probes are small DNA or RNA sequences that bind to complementary sequences in your sequences cocktail. PCR probes also include a fluorescent reporter molecule and sometimes a quencher.
Usually, PCR probes tend to be confused with primers. Although PCR probes and primers are both small sequences of DNA/RNA, a PCR probe also has a reporter. Furthermore, the use for each one is different.
While a PCR probe identifies target sequences, the primer serves as a starting point for DNA synthesis and replication. PCR probes and primers also differ in the length of their sequences and the type of DNA they bind.
For instance, a PCR probe hybridizes with double-stranded DNA, whereas a primer hybridizes with single-stranded DNA.
Another difference is that the PCR probe is between 25-1000 bp while the primer is quite small, between 18-22 bp. However, there are some systems like the Amplifluor® assay where both can be used simultaneously.
Types of PCR probes.”
With the different probes commercially available, we’re providing some tips to help you decide.
Consider that there are some PCR probes more specialized than others. For instance, hydrolysis probes using Taq polymerase are widespread and relatively cheaper than the other probes. Also, some probes have more components, or specialized components (such as enzymes), but this special feature will add to the cost.
Some probes are more difficult to design primers for. For instance, in beacon probes, the primer containing a hairpin loop structure should be carefully designed in order to make the binding between the reporter and the quencher strong enough. The trial and error could increase the cost.
Suppose you and your labmate are more familiar with hydrolysis probes. You know the protocols, you know the mechanisms, and you have a good sense of troubleshooting. In that case, it's usually safer to stick with what you know.
And in the event, you have a question, it will be a lot easier to consult your colleagues if everyone is familiar with the probe in question. That said, cost, time and other factors may still influence your decision.
Some probes are more specialized according to the application. For instance, if you are planning to use probes in gene expression validation using microarray, it is advisable to use dual hybridization and hydrolysis probes.
For pathogen detection and SNP detection, Scorpion probes are ideal.
For PCR multiplexing many other probes are better than hydrolysis probes.
We’ve put together an easy questionnaire you can use to evaluate which probe is best for your current work. Some of the sections are optional.
After choosing your probe, you’ll want to make sure your PCR conditions are optimized. Things to consider when optimizing PCR conditions are:
- Where your probe is located in relation to your primer
- Melting temperature
- Annealing temperature
- GC content
- Primer design
When designing your probe, you want to ensure that probe hybridizes close to the reverse or forward primer but does not overlap the primer. Generally, probes hybridize with either strand of the DNA duplex.
The melting temperature favors the separation of the two DNA strands of the target sequence. To improve the probe attachment to the DNA strands, the melting temperature should be 6-8 °C higher than that of the primers.
The annealing temperature is the temperature where primers successfully bind. The annealing temperature of the experiment should be 5 °C below the melting temperature of primers.
Higher GC content has higher thermal stability while lower GC content has low thermostability. If the target sequence has a high GC content, it would be harder to separate the two DNA strands using the standard temperature, and the optimal temperature for the probes will not work correctly. Therefore, the GC content of the probe should be 35-65%. The 5′ end of the probe should not contain a G to favor its binding.
Consider that the success of most of these PCR probes is related to the primer design complementary to your target sequence. You must analyze the target sequence before proceeding with the primer design. Check the GC content, probably secondary (hairpin-loops) structure formation if you work with RNA, among other factors.
Because PCR probes help locate a target sequence among many, PCR probes can be used for
In qPCR experiments, probes contribute to validation in silico data such as RNA-Seq, for pathogen detection, gene expression, and single nucleotide polymorphism detection.In-situ hybridization:
In-situ hybridization is a reaction that allows researchers to locate a target sequence in a tissue. The tissue is prepared to be mixed with the probe and allow the hybridization. Then, the tissue can be observed under a fluorescent microscope.
Probes used in real-time PCR have two main advantages.
PCR probes are designed harboring a primer that is complementary to the target sequence. Furthermore, the binding between the probe and the target sequence allows the fluorophore to light up and facilitate the quantification in real-time. This specificity favors the location of target sequences in in-situ hybridization experiments.
Due to the probe’s specificity, it is possible to target multiple sequences in the same experiment using different probes. It saves a lot of time!
PCR probe, fluorescence, PCR probe applications, PCR probe advantages.
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