The main difference between PCR primers and sequencing primers is that the PCR primers are important for PCR amplification to obtain an amplicon, whereas the sequencing primers are important for sequencing a DNA fragment to reveal its nucleotide sequence. Furthermore, two PCR primers; the forward and reverse primer are used in a PCR while sequencing requires a single sequencing primer. Moreover, PCR primers are specific to the sequence to be amplified while sequencing primers are not always related to the target DNA sequence.
PCR primers and sequencing primers are two types of short, oligonucleotide sequences responsible for helping the initiation of DNA synthesis in vitro.
Key Areas Covered
1. What are PCR Primers
– Definition, Features, Function
2. What are Sequencing Primers
– Definition, Features, Function
3. What are the Similarities Between PCR Primers and Sequencing Primers
– Outline of Common Features
4. What is the Difference Between PCR Primers and Sequencing Primers
– Comparison of Key Differences
Key Terms
Forward Primer, PCR Primers, Reverse Primer, Sequencing Primers 
What are PCR Primers
PCR primers are short DNA sequences responsible for facilitating the initiation of DNA synthesis in vitro in a process called polymerase chain reaction (PCR). Generally, DNA polymerase is the enzyme responsible for the synthesis of DNA. However, it requires a 3′ OH end for the initiation of DNA replication. Thus, the RNA primase is the enzyme that synthesizes in vivo a short RNA primer at the point of DNA replication starting site. Meanwhile, during the in vitro DNA synthesis in PCR, DNA primers are used.
Types
Furthermore, there are two types of primers used for a particular PCR reaction. They are the forward and reverse primers. Typically, forward primer anneals the antisense strand of double-stranded DNA. In comparison, reverse primer anneals to the sense strand. In general, antisense strand runs in the 3′ to 5′ direction while sense strand runs in the 5′ to 3′ direction. However, forward and the reverse primer flank the target DNA sequence to be amplified.
Figure 1: Forward and Reverse Primers
Primer Design
PCR primer design is a crucial step for a successful PCR reaction, which amplifies the target DNA sequence. Basically, the length of the PCR primers should be 18-22 bases. Also, their GC content should be 50-55%. In addition to these, the primers should have a GC-lock on the 3′ end. Furthermore, the melting temperature of primers should be 50-55 °C. Besides, poly base regions, secondary structures, and primer-dimer formation should not occur in primers.
What are Sequencing Primers
Sequencing primers are the primers, which facilitates the initiation of the sequencing reaction. Generally, both Sanger sequencing, as well as the “Next-Gen” DNA sequencing, require primers. For instance, there are two complementary DNA strands per DNA molecule. Therefore, for a particular DNA fragment, there are two sequences; sense and antisense sequence. Still, during a sequencing reaction, only a single DNA strand undergoes sequencing. Thereby, only one primer is used for the sequencing reaction. However, this primer can be either the forward primer or the reverse primer.
Figure 2: Role of Sequencing Primers in Sanger Sequencing
Moreover, one can sequence both strands of the DNA fragment in two separate sequencing reactions using forward primer and reverse primer separately in one of the two sequencing reactions. Also, sequencing primers do not necessarily need to flank the target DNA sequence as done by the forward and reverse primers. That means; sequencing primers can also anneal to the sequences in the vector backbone as well. Some of the examples of universal primers on the vector backbone for sequencing include T7, SP6, M13 reverse, CMV forward, etc.
Similarities Between PCR Primers and Sequencing Primers
- PCR primers and sequencing primers are two types of primers.
- Both are short oligonucleotide sequences.
- They are responsible for the initiation of DNA synthesis in vitro by binding to the complementary sequences of DNA.
Difference Between PCR Primers and Sequencing Primers
Definition
PCR primers refer to the short pieces of single-stranded DNA used in PCR reaction while sequencing primers refer to the short nucleotide sequences used to initiate DNA synthesis in a sequencing reaction.
Main Purpose
PCR primers are used in PCR amplification to obtain an amplicon while sequencing primers are used for sequencing a DNA fragment to reveal its nucleotide sequence.
Types
Two PCR primers; forward and reverse primer are used in a PCR while sequencing requires a single sequencing primer.
Specificity
PCR primers are specific to the sequence to be amplified while sequencing primers are not always related to the target DNA sequence.
Restriction Sites
PCR primers may contain restriction sites while sequencing primers do not contain restriction sites.
Degeneracy
PCR primers can be degenerate primers while sequencing primers are not degenerate.
Conclusion
PCR primers are the two types of primers used in the PCR reaction for the amplification of a target DNA sequence. Also, the two types of PCR primers include the forward and reverse primers. Significantly, the two primers flank the target DNA sequence, facilitating the initiation of the synthesis of each DNA strand. In contrast, sequencing primers are the primers, which help to initiate the synthesis of a complementary DNA strand during the sequencing reaction. However, only a single primer is used in the sequencing reaction. Therefore, the main difference between PCR primers and sequencing primers is their purpose and usage.
References:
1. “Types of PCR Primers.” PCR Knowledge, 1 May 2018, Available Here.
2. “PCR Primer Design Guidelines.” PRIMER Biosoft, Available Here.
3. “Sequencing Primers.” Addgene, Available Here.
Image Courtesy:
1. “Primers RevComp” By Zephyris – Own work (CC BY-SA 3.0) via Commons Wikimedia
2. “Sanger-sequencing” By Estevezj – Own work (CC BY-SA 3.0) via Commons Wikimedia
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