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Which Detection Chemistry is Right for Your qPCR Assay?

Two major types of chemistries may be utilized to detect the products of your qPCR cycles, each with particular advantages. Understanding the differences between the two will ensure you choose the most effective detection chemistry for the particular assay you are designing.

Dye Chemistry Basics

Dyes that are used for qPCR molecule detection are small molecules with high binding affinity to any double-stranded DNA, including the original double-stranded DNA present in the sample and the new copies of double-stranded DNA being formed by the PCR cycles, using various binding methods. When the dye binds to double-stranded DNA, this causes an increase in the fluorescence, and the more DNA the dye binds to, the greater the intensity of the fluorescence. The intensity of the fluorescence by the dye increases proportionally to the increase in the amount of PCR products.

Dye Advantages & Disadvantages

The main advantage of using a dye for your assay is the reduced monetary and time investment required upfront, in order to get your assay up and running. Only a simple assay design is necessary, and no time needs to be spent synthesizing one or multiple probes. Additionally, double-stranded DNA binding dyes are useful if you plan to perform post-PCR analysis, such as high-resolution melting (HRM) analysis, However, since the dye will bind to any double-stranded DNA sequence, drawbacks that come with using a dye for your assay include possible false positive signals and overall decreased assay specificity.

+ Lower costs

+ Suitable for any double-stranded DNA sequence

+ No probe synthesis

+ HRM analysis

- Decreased specificity

Probe Chemistry Basics

The more specific method of detection involves the use of an oligonucleotide probe, which contains an additional fluorescent molecule, or fluorophore, attached to it. Two types of these fluorophores exist: reporter fluorophores and quencher fluorophores. The reporter fluorophore is found on the 5’ end of the oligonucleotide and the quencher fluorophore is found on the 3’ end of the oligonucleotide. When the reporter fluorophore initially absorbs energy from light, it enters an excited state. As it returns back down to its ground state, the energy is released as fluorescence, which is then transferred to the quencher fluorophore, a process known as Fluorescence Resonance Energy Transfer (FRET). This means fluorescence intensity emitted by the reporter fluorophore is greatly reduced when the quencher fluorophore is nearby, thereby making detection of the fluorescence difficult.

One of the most common types of qPCR probes are known as hydrolysis probes. When the reporter fluorophore of the hydrolysis probe becomes separated from the quencher fluorophore, such as via cleavage from DNA polymerase 5' to 3' exonuclease activity, the intensity of the fluorescence detected from the reporter dye will increase. As the amount of amplified product increases and additional probes are cleaved, the fluorescent signal measured will increase proportionally.

In addition to hydrolysis probes, there are other probe chemistries that produce the amplified fluorescent signal, such as Molecular Beacon probes and Scorpion primer-probes.

Probe Advantages & Disadvantages

In contrast to dye chemistry, one of the main benefits gained from utilizing probe chemistry is the increased specificity of your assay through the use of an oligonucleotide probe or primer. By using fluorophores attached to the specific oligonucleotide sequence, only the specific desired PCR products will be detected, and amplification of non-targeted products will be prevented.

The other major benefit is the possibility for optical multiplexing, or amplification and detection of multiple sequences within the same reaction. This capability is not achievable through the use of dye chemistry. Multiple probes are used, each labeled with a fluorophore. If the fluorescent signal generated by each fluorophore is detected in a different optical channel, detection of each distinct target sequence is possible. If fluorophores which all produce signals within the same optical channel are attached to multiple probes with different target sequences, multiple sequences may be detected at once, but differentiation between them is not possible.

+ Higher binding specificity

+ Multiple sequences may be amplified at once and distinguished by using different optical channels

- Different probes must be synthesized for different target sequences

Dye or Probe?

In general, choosing to use a dye for your assay may make more sense to you, if you’re looking to meet these conditions:

  • More economical
  • Wide applicability
  • Design simplicity
  • Decreased set-up time
  • Post-PCR analysis

Conversely, using a probe for your assay may be the right choice, if the following criteria meet your needs:

  • Amplification of multiple target sequences
  • Detection of specific amplified products
  • Elimination of post-PCR processing

At the end of the day, there isn’t a “better” choice when it comes to detection chemistry options. It depends on the priorities specific to your assay design, and the resources you have available. Factors such as cost, number of reactions and specificity requirements are important to consider when deciding which detection chemistry to use.