Primer Dimers: A Comprehensive Guide

What are Primer Dimers?

Fluorescent real-time quantitative polymerase chain reaction (qPCR) technology is arguably the most important and widely used nucleic acid quantification technique in modern molecular biology. Successful qPCR experiments rely on precise experimental design and operational procedures. One of the most common issues encountered is the formation of primer dimers. When there are partially homologous sequences between primers, primer dimers can form. If primer dimers form during the PCR reaction, Taq DNA polymerase can extend these dimers, resulting in products longer than the original primers, which can lead to increased annealing errors during the cycling process.

Effect of Primer Dimers

The impact of primer dimers on the reaction largely depends on the chemicals used in the reaction. In reactions based on fluorescent probes, the effect of primer dimers is not significant because there is minimal occurrence of probe annealing and cleavage in the primer dimer region. In this case, the primary concern is the competitive binding of primers. However, reactions based on double-stranded DNA binding dyes are significantly affected by primer dimers. This is because the binding of dyes is non-specific, leading to enhanced fluorescence signals detected during the reaction. Consequently, this can alter the Ct values, resulting in biased outcomes.

The primer dimer in PCR. (Andrew. D. J.; et al, 2019)

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How to Determine the Presence of Primer Dimers?

Gel electrophoresis is an excellent method for visualizing primer dimers. Primer dimers form diffuse bands at the bottom of the gel, typically below 100 bp. However, gel electrophoresis alone may have limitations in sensitivity, with detection levels reaching only nanogram levels, potentially yielding inconclusive results.

Another method for detecting primer dimers is through melt curve analysis. If amplification is highly specific, the dissociation curve for each reaction well on the real-time fluorescence quantitative PCR plate will exhibit a narrow single peak. Primer dimers, characterized by lower fluorescence intensity, will display broader "waveforms" during melting at around 70°C.

What Causes Primer Dimers?

Complementary Binding of Bases at the 3' end between Primers or within Primers Themselves

The fundamental reason for primer dimer formation lies in the complementary binding of bases at the 3' end between primers or within primers themselves, allowing them to bind to each other and form double-stranded DNA fragments.

Low Template Concentration

When the template concentration is too low in PCR reactions, the chance of primer binding to the template decreases, while the possibility of interaction between primers increases. Therefore, the formation rate of primer dimers increases.

High Primer Concentration

Excessive primer concentration increases the likelihood of interaction between primers, especially in the early stages of the reaction. This may lead to excessive formation of primer dimers.

Short Primer Length

Shorter primer lengths mean fewer specific binding sites, increasing the possibility of binding between primers themselves or with other primers, especially at the 3' end.

Low Annealing Temperature

A low annealing temperature increases the chance of interaction between primers, particularly in the early stages of the reaction. This may promote the formation of primer dimers, especially before primer binding to the template.

Excessive Cycle Numbers

A higher number of PCR cycles increases the opportunity for primer interactions, especially when primer dimers have already formed. High cycle numbers may also increase the accumulation of primer dimers and elevate the risk of non-specific primer binding in PCR reactions.

How to Distinguish Between Primer Dimers and Target Bands?

To distinguish between primer dimers and target bands in experiments, the following steps are typically taken:

• Add Controls: Introducing appropriate controls is crucial in gel electrophoresis experiments. This includes replacing the template with water while keeping other conditions constant. If bands appear in certain lanes but not in control lanes, it indicates the presence of primer dimers. This control helps to eliminate the possibility of template contamination, ensuring that observed bands are formed by primer reactions.
• Understand the Nature of Primer Dimers Formation: Primer dimers form due to partial complementary binding between primers or within the 3' end region of individual primers. Therefore, primer dimer formation does not require the involvement of the template. Understanding this helps in comprehending the mechanism of primer dimer formation and provides a basis for correctly interpreting experimental results.
• Observe Band Molecular Weights: In gel electrophoresis experiments, if the expected molecular weight of the target product is small, and larger bands are observed in some lanes, it may indicate primer dimers rather than the target product. Therefore, by comparing the expected molecular weight of the target product with the observed band's molecular weight, one can preliminarily determine the presence of primer dimers.
• Differentiate from Template: When analyzing bands, it is essential to differentiate them from the template. Ensure that observed bands do not originate from template contamination. Using water instead of the template as a control helps to exclude the influence of the template on experimental results, further ensuring that observed bands are formed by primer reactions.

By following these steps, primer dimers and target bands can be effectively distinguished, accurately determining experimental results and providing a reliable basis for subsequent experiments and analysis.

How to Prevent Primer-Dimer Formation?

To prevent primer-dimer formation, various methods can be employed:

• Physical-Chemical Optimization of the PCR System: By adjusting parameters such as primer concentrations, magnesium ions, nucleotides, ionic strength, and reaction temperature, the PCR system can be optimized to reduce primer-dimer formation. However, this method is constrained by the efficiency of target sequence amplification in PCR, as reducing primer-dimer formation may lead to decreased PCR efficiency.
• Primer Design Software: Using primer design software, algorithms are utilized to assess the potential for DNA secondary structure formation and primer annealing, either to themselves or to other primers. Physical parameters considered by the software include primer self-complementarity, GC content, melting temperatures, and the absence of secondary structures in the DNA target sequence.
• Hot-Start PCR: Hot-start PCR techniques delay primer-dimer formation by inhibiting the activity of DNA polymerase until the reaction mixture reaches higher temperatures. This can be achieved through methods such as wax barriers, slow release of magnesium ions, or non-covalent binding of inhibitors.
• Primer Structural Modification: Modifying primer structures prevents extension when primers anneal to themselves or to each other. Examples include adding complementary nucleotide tails to the 5' end of primers using methods like HANDS, or using chimeric primers with RNA bases substituted for DNA bases.

By employing these methods, primer-dimer formation can be effectively prevented or reduced, thereby improving the specificity and efficiency of PCR.

How to Eliminate Primer-Dimer Formation

Methods to eliminate primer-dimer formation can be summarized as follows:

Primer Design

Consideration of primer-dimer formation should begin at the primer design stage. Avoid designing complementary sequences at the 3' end of the primer, and ensure the appropriate length of the primer. Utilize primer design software to predict and avoid dimer formation. Primer design software typically includes features to check for dimer formation, which can assess interactions between primers and avoid designing primers with complementary sequences.

Template Preparation

Ensure the quality and stability of the template, eliminating template errors that may lead to primer-dimer formation, such as impurities, deletions, or denaturation. Increasing the template concentration appropriately can help improve the efficiency of the PCR reaction. It is advisable to use methods such as internal reference genes to verify the correctness and stability of the template.

Optimization of Reaction System

Avoid Taq polymerase inactivation, paying attention to the operational details when preparing the PCR reaction system, including preparation on ice, adding Taq polymerase last, and avoiding prolonged storage of PCR products at room temperature. Ensure the correct order of addition of all reaction components. Appropriately reduce the concentrations of enzymes, primers, and magnesium ions. Experimental trials with a gradient of primer concentrations can be conducted to find the optimal concentration combination. Experimental trials can be performed to determine the best reaction conditions.

Optimization of Reaction Conditions

Design a gradient of annealing temperatures and find the most suitable annealing temperature to prevent primer-dimer formation. Typically, increasing the annealing temperature can effectively solve the problem. Different annealing temperatures can be tried in experiments to find the optimal conditions. Appropriately reduce the number of PCR cycles, generally to 30-40 cycles, to avoid primer mismatches and enzyme efficiency reduction. By optimizing the number of PCR cycles, the risk of primer-dimer formation can be minimized.

Reference

1. Andrew. D. J.; et al. PrimerROC: Accurate Condition-Independent Dimer Prediction Using ROC Analysis. Scientific Reports. 2019, 9: 209.