What is the Difference Between Forward and Reverse Primers

The main difference between forward and reverse primers is that forward primers anneal to the antisense strand of the double-stranded DNA, which runs from 3′ to 5′ direction, whereas reverse primers anneal to the sense strand of the double-stranded DNA, which runs from 5′ to 3′ direction. Furthermore, 5′ primers refer to forward primers, while 3′ primers refer to reverse primers. 

Forward and reverse primers are the two types of primers used in the PCR (polymerase chain reaction) to amplify a specific part of a DNA strand. 

Key Areas Covered 

1. What are Forward Primers
     – Definition, Features, Importance
2. What are Reverse Primers
     – Definition, Features, Importance
3. What are the Similarities Between Forward and Reverse Primers
     – Outline of Common Features
4. What is the Difference Between Forward and Reverse Primers
     – Comparison of Key Differences

Key Terms 

3′ Primers, 5′ Primers, Antisense Strand, Forward Primers, PCR, Reverse Primers, Sense Strand

What are Forward Primers 

Forward primers are one of the two types of primers used in a PCR setup. The main significant feature of forward primers is that they anneal to the antisense or (-) strand of the double-stranded DNA. Generally, the antisense strand serves as the template strand for the synthesis of mRNA. Therefore, this strand is also known as the coding strand. 

Figure 1: Primer Annealing

However, the main function of the forward primers is to amplify the antisense strands during PCR.

What are Reverse Primers  

Reverse primers are the second type of primers used in the PCR setup. They anneal to the sense or the (+) strand of the double-stranded DNA. The sense strand is complementary to the template strand and therefore, it is known as the anticoding strand. 

Figure 2: Primer Function

Moreover, the main function of the reverse primers is to amplify sense strands during PCR.

Similarities Between Forward and Reverse Primers 

• Forward and reverse primers are two types of primers that are useful in PCR. 
• Both are oligonucleotides used for the initiation of PCR. 
• Also, their length varies between 18 to 25 base pairs. 
• Additionally, they run in the 5′ to 3′ direction from left to the right.  
• Besides, they are complementary DNA, which anneals to the single-stranded DNA during the annealing step. 
• Moreover, their annealing occurs at higher temperatures and their melting temperatures (Tm) should be within 55°C and 65°C. 
• Importantly, the maximum difference between the melting temperatures of both primers should be  5°C.  
• Also, their GC content should be between 40 and 60%, with the 3′ of a primer ending in C or G to promote binding.  
• They should not contain regions forming secondary structures. 
• Furthermore, they should avoid self-dimers/hairpins and primer-dimer formation. 

Difference Between Forward and Reverse Primers 

Definition 

Forward primers refer to the PCR primers, which are complementary to the antisense strand of double-stranded DNA, while reverse primers refer to the PCR primers, which are complementary to the sense strand of the double-stranded DNA. Thus, this is the main difference between forward and reverse primers.

Also Known as 

5′ primers refer to forward primers, while 3′ primers refer to reverse primers. 

Function 

Moreover, forward primers are responsible for the amplification of the antisense strand, while reverse primers are responsible for the amplification of the sense strand. 

Occurrence 

Also, another difference between forward and reverse primers is that forward primers occur at the 5′ end of the PCR product, while reverse primers occur at the 3′ end of the PCR product. 

Conclusion 

Forward primers are one of the two primers used in the PCR. Moreover, they anneal to the antisense strand of DNA. In contrast, reverse primers are the second type of primers used in PCR. They anneal to the sense strand of DNA. The main function of them is to amplify a specific piece of DNA. However, the main difference between forward and reverse primers is the type of DNA strand to which they anneal. 

References:

1. “Polymerase Chain Reaction (PCR).” Diamantina Institute, The University of Queensland, 9 Feb. 2018, Available Here.

Image Courtesy:

1. “Primers RevComp Melted2″By Richard Wheeler (Zephyris) – Own work (CC BY-SA 3.0) via Commons Wikimedia  
2. “Primers RevComp Elongation2” By Richard Wheeler (Zephyris) – Own work (CC BY-SA 3.0) via Commons Wikimedia