How to Simplify PCR Optimization Steps for Primer Annealing

Introducing a universal annealing temperature for primers

Primer annealing is a critical step in polymerase chain reaction or PCR. In this step, the primers bind to flanking sequences of the target DNA for amplification. The annealing temperature of this step should be determined from the melting temperature of the selected primers to help ensure specificity of primer binding and target amplification. (Learn more: PCR setup and cycling parameters)

What are some challenges in the annealing step of PCR?

The recommended melting temperature of PCR primers is usually in the range of 55°C to 70°C and within 5°C of each other. Because of the differences in sequence, length, and composition of the primers, it is often difficult to have similar melting temperatures (T_ms) between the two. In that case, the primer with the higher T_m could bind to unintended targets, while the primer with the lower T_m would have difficulty binding at an annealing temperature chosen for these primers (Figure 1). This can drastically reduce the yield and specificity of PCR and even cause PCR to fail.

Annealing of PCR primers at 60°C under suboptimal conditions

Figure 1. Suboptimal binding of primers at 60°C without optimization. In typical PCR, the primer with T_m >60°C could bind to unintended targets whereas the primer with T_m <60°C could only bind partially or remain unbound.

To enable PCR success with maximal yield and specificity, the annealing temperature of each primer set and its target should be optimized (Figure 2). This optimization process can be long and tedious, especially when amplifying multiple DNA targets using different primer sets.

PCR gel with results of annealing temperature optimization

Figure 2. PCR optimization by annealing temperatures. The annealing temperature was optimized in a gradient thermal cycler by increasing the temperature in 2-degree steps. The optimal annealing temperature in this case was 56°C.

How can I overcome challenges associated with PCR annealing?

To help simplify and save time in PCR, we (Thermo Fisher Scientific) have developed novel Invitrogen Platinum DNA polymerases with reaction buffers that allow a universal annealing temperature of 60°C. Their buffers are designed with an isostabilizing component, which increases the stability of primer-template duplexes during the annealing step (Figure 3).

Annealing of PCR primers under universal annealing condition

Figure 3. Primer binding in Platinum PCR products with the universal annealing buffer. The isostabilizing component enables specific binding of the primers to the DNA template, even when their melting temperatures differ from the 60°C annealing temperature.

This innovation of the universal annealing buffer enables you to circumvent calculation of the annealing temperature of each primer set, without compromising yield and specificity of PCR (Figure 4).

Gel of PCR products amplified with a universal annealing temperature of 60°C

Figure 4.PCR amplification with high specificity and yield using a universal annealing temperature of 60°C. Primer sets with a range of annealing temperatures were used to amplify 12 targets in human genomic DNA with a 60°C annealing temperature using Platinum SuperFi II DNA Polymerase. The annealing temperatures stated were calculated using the T_m calculator for Platinum SuperFi DNA Polymerase. Molecular weight marker (M): Invitrogen TrackIt 1 Kb Plus DNA Ladder.

Is there more I can expect from the universal annealing?

We’re glad you asked! The innovative feature of the universal annealing also allows cycling of shorter and longer amplicons together. Typically, amplification of different target lengths needs varying extension times, because lengthy extension times would often lead to nonspecific amplification. However, the isostabilizing component of the universal annealing buffer offers stability of primer-template duplexes. This enables amplification of different lengths using the same extension time selected for the longest amplicons, without compromising specificity (Figure 5).

Gels of PCR amplicons from long extension times with or without universal annealing

Figure 5. The universal annealing feature enables co-cycling of short and long amplicons. Four targets of different lengths were amplified from human genomic DNA using Platinum II Taq Hot-Start DNA Polymerase (with the universal annealing feature) or another hot-start DNA polymerase (without the universal annealing feature). The same PCR protocol was used for all four targets with the annealing and extension settings indicated. Molecular weight marker: Thermo Scientific ZipRuler Express DNA Ladder 2.

In this way, different PCR assays can be co-cycled using the same protocol, instead of sequential runs, with the universal annealing buffer (Figure 6). This can help you save significant time and simplify PCR protocols, especially when you need different primer sets to detect multiple sequences.

PCR amplification workflows with and without co-cycling capability

Figure 6. Time saving enabled by assay co-cycling and a universal protocol. PCR assays using conventional protocols require sequential runs for amplification of targets of different lengths because of the different primer annealing temperatures and extension steps. With Platinum DNA Polymerases with the universal annealing, different PCR assays can be cycled together using a universal protocol with one primer annealing temperature (60°C) and the extension time selected for the longest amplicon.

Can I try the universal annealing in my PCR?

Our latest Platinum DNA polymerases are designed for a universal annealing temperature of 60°C. Request a sample (where available) to try them for free in your experiments. These products can help you save time and simplify PCR protocols by:

Reducing the need for optimization of primer annealing,
Enabling primer–template annealing at 60°C, and
Allowing co-cycling of different PCR targets.

Ultrahigh fidelity for highest confidence in PCR sequence accuracy

Platinum SuperFi II DNA Polymerase

Engineered enzyme for high specificity and yield with fast cycling

Platinum II Taq Hot-Start DNA Polymerase

Universal master mix for a direct route to PCR

Platinum Direct PCR Universal Master Mix

Benefits of a universal annealing temperature for PCR primers

Learn the importance of the annealing step in PCR, how to circumvent optimization steps using a specially formulated PCR buffer, and the benefits of a universal annealing temperature enabled by the buffer.