Primers in PCR
A primer is a small single-stranded DNA molecule that participates in the PCR reaction and provides a free 3′ hydroxyl end for the Taq polymerase enzyme. In complex organisms like humans, only one of the two DNA strands functions as the active gene in any given region. The usual goal of performing PCR is to amplify this region, referred to as the Target Region. For this purpose, two primers are required; one binds to the target strand and the other to its complementary strand.
These two primers are called the Forward and Reverse primers, represented by the abbreviations F and R. The binding of primers to the template molecule is based on hydrogen bonds, where the 5′ end of the primer attaches to the 3′ end of the template molecule.
Primer Binding
In any given region, only one of the two DNA strands contains the active gene. This strand is referred to as the Anti-Sense strand, and its sequence is, in fact, the reverse of what is recorded in databases as the gene sequence. According to a global convention, all databases provide the transcript sequence of the gene—not the gene itself. This is why in all these databases, the start codon of all protein-coding sequences is ATG, which is directly translated to methionine. This sequence is actually the AUG found in mRNA, transcribed from its complement (i.e., TAC) in the anti-sense DNA.
The other DNA strand, in which no coding sequence is observed in the target region, is called the Sense strand. It is named so because its sequence, in the 5′ to 3′ direction, exactly matches the mRNA transcript. The forward primer must bind to the anti-sense strand, so it is designed to ensure that the 5′ end nucleotide is complementary to the nucleotide at the 3′ end of the target region in the anti-sense strand.
Thus, if we move along the primer and transcript strands—whose sequence is accepted as the gene—in the 5′ to 3′ direction, we will encounter the same sequence of nucleotides. For this reason, this primer is called the Forward primer. The same applies to the reverse primer; this primer binds to the opposite DNA strand (the sense strand), and its sequence, in the 5′ to 3′ direction, is the reverse and complement of the target (gene).
Key Terms for Primer Design
Before examining the principles of primer design, two scientific terms should be familiarized:
Efficiency:
This term describes how well the primer binds to its complementary sequence in the target region and successfully generates the final product. High efficiency of a primer pair results in a significant amount of product and consequently a very strong band in gel electrophoresis.
Specificity:
This term describes how well the enzyme can bind only to its complementary sequence in the target region and not to any other point on the template strand. Greater specificity of a primer pair means a reduction in non-specific bands in gel electrophoresis.
Maintaining both of these characteristics in a primer pair can sometimes be challenging, as increasing one often requires reducing the other. Additionally, factors such as annealing temperature and GC content must be considered in primer design, making the process complex yet essential.
Factors Affecting Primer Efficiency and Specificity
Factors that influence the efficiency and specificity of primers include:
Primer length
Melting Temperature (Tm)
Primer Annealing Temperature
GC Content along the primer sequence
GC Clamp
Secondary Structures
Repeats
Runs of consecutive nucleotides
3′ Stability
Prevention of Secondary Structure Formation in the Template Molecule
Primer Length
Generally, the specificity of a primer is adjusted by changing the length of this molecule, which in turn affects the annealing temperature. In most cases, primers that are 18 to 24 nucleotides long have high specificity, and within this range, increasing each nucleotide enhances the primer's specificity fourfold. Reducing the length of these strands significantly impacts their ability to bind specifically, such that primers shorter than 15 nucleotides tend to bind to nearly any point in the genome.
Although this feature can make them ineffective in standard PCRs, this random binding creates opportunities for their use in specific reactions, such as Arbitrary PCR, which is employed in genomic mapping of simple organisms.
While there is no limit to increasing the length of primers, it is essential to note that longer primers will increase the annealing temperature and time, as well as the likelihood of secondary structure and dimer formation.
In general, primers that are 28 to 35 nucleotides long are used only when the template shows levels of heterogeneity, or when the researcher wishes to attach a specific sequence—such as an enzyme cut site—to it. It is evident that in this case, a portion of the primer's sequence will not bind to the template during the first round of PCR. To address this issue, a lower annealing temperature is considered in the initial rounds, which is then gradually increased to the permissible level in subsequent rounds.
Software for Primer Design
Today, various software programs for primer design are available that can significantly reduce time, cost, and labor. A noteworthy point regarding the use of these software tools is the variability in their output results. This can be somewhat confusing, especially for less experienced researchers.
It is important to note that this variability is often due to the algorithms these programs use, and sometimes, by changing some default settings, you can achieve consistent results. Some commonly used software for primer design include:
Oligo:
One of the most widely used and user-friendly primer design software. This program allows researchers to design, analyze, and compare a pair of primers or a set of them. These analyses include examining various features of the proposed primers, including secondary structures, false priming sites, internal stability, and some physical characteristics. With internet access, Oligo can fetch and store some of its required data directly from the NCBI database.
Primer Premier:
This software automatically implements all necessary guidelines for designing a good primer pair. With its help, you can design specific or degenerate primers for a single sequence or a set of aligned primers. This program also allows for the examination of restriction sites, analysis of contig pieces, and primer design for sequencing.
AlleleID& Beacon Designer:
These are two software tools optimized for qPCR. They are used in the design of primers and probes necessary for identifying complexes.
PrimerPlex:
This program allows you to design the primers needed for Multiplex PCR and for multiple SNP studies in genotype determination.