Difference Between PCR Primers and Sequencing Primers

Key Difference – PCR Primers vs Sequencing Primers
 

With the recent developments in the field of molecular biology, different genetic techniques were developed that made the investigation processes of different avenues of the subject easy and accurate. PCR and other sequencing procedures are two important such techniques. They use different subcomponents. Primers are considered as the major sub-component common to both PCR and Sequencing techniques. PCR primers are used for amplification of a particular DNA sequence whilst sequencing primers are used in the context of sequencing a DNA fragment with the intention of revealing its specific order of the nucleotide sequence. This is the key difference between PCR primers and sequencing primers.

CONTENTS

1. Overview and Key Difference
2. What are PCR Primers
3. What are Sequencing Primers
4. Similarities Between PCR Primers and Sequencing Primers
5. Side by Side Comparison – PCR Primers vs Sequencing Primers in Tabular Form
6. Summary

What are PCR Primers?

Polymerase Chain Reaction (PCR) is a genetic technique that is utilized in the field of molecular biology in order to amplify a single or few copies of a particular DNA segment and to obtain many millions of identical copies. In a PCR reaction, different components are used including primers. Primers are short DNA strands with a nucleotide length of 18-25 making them compatible with the start and the end region of the DNA fragments to be amplified. Primers can be a forward primer and reverse primer. These primers bind to the DNA fragment at the specific points where it makes DNA polymerase to bind to the specific primer at the location and initiate the synthesis of the new DNA strand.

The selection of primers is an important aspect of PCR process. The selection of the length of the primer is important. The ideal length would be 18-25 nucleotides. If the length is too short or too long, the primers will not bind to the DNA sequence to be amplified accurately. Primers that are too short in length leads to non-specific primer annealing at different locations of the DNA sequence.

Figure 01: PCR Primers

The Guanine and Cytosine (GC) content in a good primer should be in the range of 40-60. The primer annealing temperature and melting temperature are vital factors during PCR. The melting temperature should be calculated accurately, and the primer annealing temperature should be 5 ⁰C less than the melting temperature. The melting temperature should be 60 °C and 75 °C. Too high or too low temperatures will result in less active DNA polymerase activity.

What are Sequencing Primers?

Sequencing primers are used in the context of sequencing a DNA fragment with the intention of revealing its specific identity. To obtain good sequencing results high quality primers and templates are important. Thus, when primers are selected, they should be unique to a particular region where we desire to sequence. It also should be with a correct orientation where the sequences are usually generated from 3’ to 5’ ends of the primers. The sequence should be lacking undesirable self-hybridization such as the formation of hairpin loops. It should not contain consecutive formation of Guanine bases.

The melting temperature (Tm) of the primer must be suitable for the conditions of the sequencing. Therefore, it should lie between 52^oC and 74^oC. Preparation of oligonucleotides to be used as a primer should be purified to obtain the wanted full-length of the sequence. If the oligonucleotides contain impurities, the primer sequence signalling will be superimposed from different priming sites, and it also will decrease the number of base cells.

Figure 02: Sequencing Primers

The primer melting temperature (Tm) of an oligonucleotide determines, how strong the complementary DNA strands are hybridized with each other. Tm can be considered as a thermodynamic calculation where it is dependent on both DNA sequences and several conditions such as salt concentration. The Tm is important during PCR where a variant called the cycle sequencing is used to produce a group of dideoxynucleotide-terminated fragments. Here, the primer that is sequenced will initially be annealed alternatively, then extended and lastly denatured for amplification. Therefore, the Tm value should be in between 52^oC and 74^o C. Synthesized oligonucleotides can be obtained from DNA/RNA synthesis laboratories as per choice. The small scale of synthesis that is used for DNA sequencing is usually 50 nmol. Also most importantly the primers used for sequencing should be purified to be free of impurities that will prevent the quality reduction.

What are the Similarities Between PCR Primers and Sequencing Primers?

• Both PCR Primers and Sequencing Primers are primers that are used in the amplification process of a targeted DNA sequence.
• Both PCR Primers and Sequencing Primers are composed of nucleotides.
• Both PCR Primers and Sequencing Primers are short oligomers.

What is the Difference Between PCR Primers and Sequencing Primers?

Summary – PCR Primers vs Sequencing Primers

Sequencing primers are used in the context of sequencing a DNA fragment with the intention of revealing its specific identity. One sequencing primer will be enough to run the process. To obtain good sequencing results, high quality primers and templates are important. Thus, when primers are selected, they should be unique to a particular region where we desire to sequence. PCR Primers are short DNA strands with a nucleotide length of 18-25 which is compatible with the start and the end region of the DNA fragments that are to be amplified. PCR primers can be a forward primer and reverse primer. The Guanine and Cytosine (GC) content in a good primer should be in the range of 40-60. The primer annealing temperature and melting temperature are vital aspects during PCR. This is the difference between PCR primers and Sequencing primers.

Reference:

1.“Polymerase chain reaction (PCR).” Khan Academy. Available here
2.“Sequencing primers and primer design.” Sequencing primers and primer design | University Core DNA Services | University of Calgary. Available here    

Image Courtesy:

1.’Primers RevComp’By Zephyris – Own work, (CC BY-SA 3.0) via Commons Wikimedia 
2.’DNA Sequencin 3 labeling methods’By Abizar (original uploader)  at English Wikipedia – Transferred  by Gustavocarra., (Public Domain) via Commons Wikimedia

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