Difference Between Probe and Primer

Main Difference – Probe vs Primer

PCR is a technique used in biotechnology to amplify specific DNA fragments for various purposes. Probe and primer are two types of single-stranded, oligonucleotides used in various types of PCR. Probes are mainly used in qPCR while synthetic primers are used in every type of PCR. The main difference between probe and primer is that probe is that probe is used to detect the presence of a specific DNA fragment in the mixture through the hybridization with a double-stranded DNA whereas primer is used in the initiation of the polymerase chain reaction by hybridization with single-stranded DNA. Generally, primers are used in the initiation of DNA replication inside the cell. Probes are used in hybridization reactions as well.

Key Areas Covered

1. What is a Probe
– Definition, Design, Importance
2. What is a Primer
– Definition, Design, Importance
3. What are the Similarities Between Probe and Primer
– Outline of Common Features
4. What is the Difference Between Probe and Primer
– Comparison of Key Differences

Key Terms: Hybridization, Oligonucleotides, PCR, Primer, Probe, QPCR

What is a Probe

Probe is a fragment of DNA or RNA used to detect the presence of a specific DNA fragment within a sample. Therefore, probes can be used for two types of techniques, in qPCR and in hybridization reactions. Four facts should be considered in the designing of a probe.

Location – The probes should hybridize with the DNA strand in a close proximity to the reverse or forward primer. But, it should not overlap with the primer binding sites. Generally, probes hybridize with either strand of the DNA duplex.
Melting temperature (Tm) – The melting temperature of the probe should be 6-8 °C higher than that of the primers.
Annealing temperature (Ta) – The annealing temperature of the experiment should be 5 °C below the melting temperature of primers.
GC content – The GC content of the probe should be 35-65%. The 5′ end of the probe should not contain a G.

In qPCR, probes are labeled with fluorescent dyes or radioactive elements. These probes are hybridized with the target sequence in the DNA duplex. Different types of labeled probes, either with radioactive elements or fluorescence, are used in various types of hybridization reactions as well. The hybridization of the PNA probes to their target sequences are shown in figure 1. PNA probes are used to determine the length of the telomeres.

Difference Between Probe and Primer - Figure 1

Figure 1: Hybridization of PNA Probes

During hybridization, probes bind to the single-stranded DNA in a complementary manner.

What is a Primer

Primer refers to a short strand of DNA or RNA that serves as the starting point of DNA synthesis. RNA primers are used inside the cell to initiate the DNA replication with the aid of DNA polymerase. Synthetic DNA primers are mostly used in PCR to amplify the desired DNA fragment. The target sequence is flanked by two primers known as forward primer and reverse primer. Specificity and complementarity are the primary factors in the designing of primers. Secondary structures should also be avoided. Other factors that should be considered during the designing of primers are described below.

Melting temperature (Tm) – The optimal melting temperature of both forward and reverse primer should be 60-64 °C.
Annealing temperature – The annealing temperature of the experiment should be 5 °C below the melting temperature of each primer.
GC content – The GC content of primers should be 35-65%.

The annealing of the forward and reverse primer to the two strands of the target DNA is shown in figure 2.

Figure 2: Primer Annealing

In DNA sequencing, primers are used in the amplification of the target fragment. Primers can be labeled either with radioactive elements or fluorescence for various detection purposes.

Similarities Between Probe and Primer

Probe and primer are two types of single-stranded, oligonucleotides used in various PCR techniques to hybridize with complementary DNA.
Both probe and primer are specific to a particular DNA fragment.
Both probe and primer can be either DNA/RNA.
Both probes and primer have specific temperatures to anneal with the target sequence.
Both probe and primer can be entangled with a fluorophore for the detection.

Definition

Probe: Probe is a fragment of DNA or RNA used to detect the presence of a specific DNA fragment within a sample.

Primer: Primer is a short strand of DNA or RNA that serves as the starting point for DNA synthesis.

Role

Probe: Probes are used to detect a specific DNA fragment in qPCR.

Primer: Primers are used to initiate the DNA replication. It is also used in the initiation of the PCR.

Length

Probe: The length of a probe can range from 25-1000 base pairs.

Primer: The length of a primer can range from 18-22 base pairs.

Hybridization

Probe: Probes are hybridized with double-stranded DNA.

Primer: Primers are hybridized with single-stranded DNA.

Labeling

Probe: Probes are generally labeled with a fluorophore for the detection.

Primer: Primers can be labeled based on the purpose.

Conclusion

Probe and primer are two types of single-stranded oligonucleotides used in various types of PCR. Probes are used in the detection of specific DNA fragments in qPCR. Primers are used to initiate DNA replication inside the cell and they are also used in the initiation of PCR. Therefore, the main difference between probe and primer is their purpose.

Reference:

1. Designing PCR primers and probes, Integrated DNA Technologies, Available here.

Image Courtesy:

1. “Q-FISH workflow” By Jclam at English Wikipedia (CC BY 3.0) via Commons Wikimedia
2. “Primers RevComp Elongation” By Richard Wheeler (Zephyris) – Own work (CC BY-SA 3.0) via Commons Wikimedia