Key Difference – Probe vs Primer
The molecular probe is a small DNA or RNA fragment that recognizes the complementary sequences in DNA or RNA and allows identification of the target sequence. Primer is a small stretch of DNA or RNA which serves as a starting point for DNA synthesis. Primers and probes hybridize with the complementary nucleotides of the template DNA or the target DNA. However, the key difference between probe and primer is that primers are necessary for DNA replication while probes are necessary for detection of specific sequences in the sample DNA.
CONTENTS
1. Overview and Key Difference
2. What is Probe
3. What is Primer
4. Side by Side Comparison – Probe vs Primer
5. Summary
What is a Probe?
Probe is a small fragment of DNA or RNA used to detect the target DNA or RNA in the sample by molecular hybridization. They are also known as molecular markers. The length of the probe can vary (100 to 1000 bases), and probe nucleotides are complementary to the part of the target sequence. For the ease of detection, probes are labeled with radioactive isotopes or with fluorescent dyes or antibodies. Probes bind with the complementary bases of the target sequence and reveal the presence of the target DNA or RNA in the sample. There are two main methods of labelling probes: end labeling and nick translation. Probes are categorized into different types including DNA probes, RNA probes, cDNa probes and synthetic oligonucleotides probes, and they are prepared using different techniques.
Probes are important tools in many microbial and molecular areas such as virology, forensic pathology, paternity testing, DNA fingerprinting, detection of genetic diseases, RFLP, molecular cytogenetics, in situ hybridization, etc.
Figure 01: Fluorescently labeled probe used in FISH for pathogen detection
What is a Primer?
Primer is a short DNA or RNA fragment which serves as an initiator for DNA synthesis. DNA polymerase enzyme adds nucleotides to the 3’ OH group of primer sequence and synthesizes the new strand complementary to the template DNA. Primers are very short fragments with the length of 18 to 20 nucleotides. They are chemically synthesized in the laboratory for in vitro DNA amplification (PCR). Primers can have any sequence of nucleotides since they are designed by the user. They are synthesized to match with the complementary bases of the template DNA. Therefore, it can have any sequence of nucleotides. Primers are of utmost importance for DNA replication since DNA polymerase can’t synthesize new DNA without a preexisting piece of DNA. When designing the primers for PCR, following things need to be considered:
- Primers should contain the complementary nucleotides to the flanking end of the DNA that wants to amplify.
- Primers should have melting temperature between 55 – 65 0C
- G and C content should be between 50 to 60%.
Two primers are used in PCR as forward and reverse to replicate both strands of the sample DNA. Primers are commonly used to perform PCR and DNA sequencing.
Figure 02: Primer annealing in PCR
What is the difference between Probe and Primer?
Probe vs Primer |
|
| Probe is a small fragment of DNA/RNA used to detect the presence of the target sequence in a sample by molecular hybridization. | Primer is a small stretch of DNA or RNA that serves as a starting point for DNA replication. |
| Function | |
| This detects the presence of a specific sequence in the sample of DNA or RNA. | This acts as a starting point for DNA synthesis. |
| Length | |
| Length can be in the range of 100 – 1000 bases | Length is generally about 18 – 20 bases |
| Binding with Complementary Sequence | |
| Probe hybridizes with the complementary bases of the target sequence | Primer anneals with the complementary bases of the DNA strands. |
| Labeling | |
| Probes are labeled for ease of detection | Primers are generally not labeled |
| Use in PCR | |
| Probes are not used in PCR | Primers are used in PCR |
Summary – Probe vs Primer
Probe is a small fragment of DNA or RNA sequence that can be hybridized with complementary nucleotides to detect a target sequence in the sample. Probes are labeled radioactively, immunologically or fluorescently to see the presence of target sequence. Primer is a very small DNA or RNA fragment that act as the starting point for in vitro DNA amplification. DNA polymerase identifies 3’ OH group primer and initiates the building of new strand complementary to the template. Probes and primers work similarly by hybridizing with complementary nucleotides. Thus, the key difference between probe and primer is their prime function.
Reference:
1.”Primer (molecular biology).” Wikipedia. Wikimedia Foundation, 02 Feb. 2017. Web. 01 Mar. 2017.
2.”Hybridization probe.” Wikipedia. Wikimedia Foundation, 30 Dec. 2016. Web. 01 Mar. 2017
3.”Primer (molecular biology).” Primer (molecular biology) – ScienceDirect Topics. N.p., n.d. Web. 01 Mar. 2017
Image Courtesy:
1. “FISH for Bacterial Pathogen Identification” By Pepetps Togopic Ivan Akira Magnus ManskeTimothy W. Ford – (CC BY-SA 3.0) via Commons Wikimedia
2. “Primers RevComp Melted2” By Richard Wheeler (Zephyris) – Own work (CC BY-SA 3.0) via Commons Wikimedia