PCR Cycling Parameters–Six Key Considerations for Success

Figure 1. Illustration of the main steps in PCR─denaturation, annealing, extension─to amplify target sequence from a template DNA.
 

The initial denaturation step is carried out at the beginning of PCR to separate the double-stranded template DNA into single strands so that the primers can bind to the target region and initiate extension. Complete denaturation of the input DNA helps ensure efficient amplification of the target sequence during the first amplification cycle. Furthermore, the high temperature at this step helps inactivate heat-labile proteases or nucleases that may be present in the sample, with minimal impact on thermostable DNA polymerases. When using a hot-start DNA polymerase, this step also serves to activate the enzyme, although a separate activation step may be recommended by the enzyme supplier.

The initial denaturation step is commonly performed at 94–98°C for 1–3 minutes. The time and temperature of this step can vary depending on the nature of the template DNA and salt concentrations of buffer. For example, mammalian genomic DNA may require longer incubation periods than plasmids and PCR products, based on DNA complexity and size. Similarly, DNA with high GC content (e.g., >65%) often calls for longer incubation or higher temperature for denaturation (Figure 2). Buffers with high salts (as required by some DNA polymerases) generally need higher denaturation temperatures (e.g., 98°C) to separate double-stranded DNA (Figure 3).

Some DNA polymerases such as Taq DNA polymerase can become easily denatured from prolonged incubation above 95°C. To compensate for decreased activity in this scenario, more enzymes may be added after the initial denaturation step, or a higher-than-recommended amount of DNA polymerase can be added at the beginning. Highly thermostable enzymes such as those derived from Archaea are able to withstand prolonged high temperatures and remain active throughout PCR (learn more about DNA polymerase characteristics).

After the initial denaturation step, subsequent PCR cycles begin with a separate denaturation step that lasts 0.5–2 minutes at 94–98°C. As with the initial template DNA denaturation step, the time and temperature should be optimized according to the nature of the template DNA, DNA polymerase, and buffer components. For instance, long and/or GC-rich DNA targets may benefit from a prolonged incubation and/or a higher temperature (Figures 2, 3). The presence of additives such as glycerol, DMSO, formamide, and betaine can enhance separation of double-stranded DNA during the denaturation step and promote specificity, overcoming a need for longer incubation or higher temperature (see reaction component considerations).

In this step, the reaction temperature is lowered to allow binding of the primers to the target DNA. Often, incubation time of 0.5–2 minutes is sufficient for primer annealing. The annealing temperature is determined by calculating the melting temperature (T_m) of the selected primers for PCR amplification. A general rule of thumb is to begin with an annealing temperature 3–5°C lower than the lowest T_m of the primers.

T_m is defined as the temperature at which 50% of the primer and its complementary sequence form a duplex, and it can be calculated in a number of ways. The simplest method in estimating primer T_m is by the number of nucleotides present in the DNA oligo, using the formula:

T_m = 4 (G + C) + 2 (A + T)

Since the salt concentration (Na⁺) of the reaction impacts primer annealing, T_m can be more accurately calculated with the formula:

T_m = 81.5 + 16.6(log[Na⁺]) + 0.41(%GC) – 675/primer length

Using thermodynamic stability of every adjacent dinucleotide pair of the oligo, in combination with concentrations of salts and primers, T_m can also be calculated with a method called the Nearest Neighbor method [1,2]. This method is also the basis of our online tool to determine primer annealing temperatures recommended for specific DNA polymerases.

One important consideration in T_m calculation is the use of PCR additives, co-solvents, and modified nucleotides. The presence of these reagents lowers the T_m of the primer-template complex. For instance, 10% DMSO can decrease the annealing temperature by 5.5–6.0°C [3]. Likewise, substitution of dGTP with 7-deaza-dGTP in PCR will also decrease the T_m. In these cases, the annealing temperature should be adjusted accordingly.

Note that the calculated T_m value is meant as a starting reference temperature for primer annealing. Annealing temperature may need further optimization, depending on the amplification results. For instance, if the results are no or low amplification, the annealing temperature may be lowered in increments of 2–3°C during optimization. If nonspecific PCR products appear, however, the annealing temperature can be raised in increments of 2–3°C (up to the extension temperature) to enhance specificity (Figure 4).

To help minimize this optimization step and save time, the reaction buffer of some DNA polymerases is designed with isostabilizing components. This special formulation increases stability of primer–template duplexes during the annealing step, thereby improving yield and enhancing specificity of PCR. In addition, the buffer enables PCR primer–template annealing at a universal temperature (e.g., 60°C), even with primers of different melting temperatures.

For optimization of annealing temperatures, gradient thermal cycler blocks are popular options, where highest and lowest temperatures are set across the block so variations in temperature can be assessed across a series of wells or reactions at the same time. In practice, a true gradient with precise temperature control of the wells is difficult to attain and “better-than-gradients” blocks with separate heating/cooling units are recommended for precise temperature control over PCR optimization (Figure 5). (Learn more: Thermal cycler considerations).

Figure 5. Comparison of block temperatures of thermal cyclers using “better-than-gradient” vs. standard gradient technologies.
 

After primer annealing, the next step in PCR is to extend the 3′ end of primers, complementary to the template. In this step, 5′→ 3′ polymerase activity of the DNA polymerase incorporates dNTPs and synthesizes the daughter strands. The reaction temperature is raised to the optimal temperature of the enzyme for its maximal activity, which is generally 70–75°C for thermostable DNA polymerases. If the primer annealing temperature is within 3°C of the extension temperature, both annealing and extension temperatures can be combined into a single step called two-step PCR, instead of conventional three-step PCR. Two-step PCR shortens the time taken for the PCR process as there is no need for switching and stabilizing temperatures between annealing and extension.

The extension time of PCR depends upon the synthesis rate of DNA polymerase and the length of target DNA. The typical extension time for Taq DNA Polymerase is 1 min/kb, whereas that of Pfu DNA polymerase is 2 min/kb. Therefore, “slow” enzymes will require more time to amplify than their “fast” counterparts for comparable yields (Figure 6). Similarly, long DNA amplicons will require longer extension times than short DNA for full-length replication. In addition to increasing extension time in amplifying long targets (e.g., >10 kb), reducing temperatures of the PCR steps may be necessary to ensure primer binding and sustained enzyme activity during prolonged cycling.

PCR steps of denaturation, annealing, and extension are repeated (or “cycled”) many times to amplify the target DNA. The number of cycles is usually carried out 25–35 times but may vary upon the amount of DNA input and the desired yield of PCR product. If the DNA input is fewer than 10 copies, up to 40 cycles may be required to produce a sufficient yield. More than 45 cycles is not recommended as nonspecific bands start to appear with higher numbers of cycles. Also, accumulation of by-products and depletion of reaction components drastically lower PCR efficiency, resulting in a characteristic plateau phase for a PCR amplification curve (Figure 7). Conversely, low cycle numbers are preferable for unbiased amplification (as in next-generation sequencing) and accurate replication of target DNA (as in cloning).

The final extension step follows completion of the last PCR cycle. In this step, the PCR mixture is incubated at the extension temperature (generally 72°C) for a final 5–15 minute period. The duration of this final step also depends on the amplicon length and composition and should be optimized to ensure full-length polymerization and good yield of the target DNA (Figure 8). In addition to filling in incomplete ends, DNA polymerases with terminal deoxynucleotide transferase activity (TdT) such as Taq DNA polymerase add extra nucleotides to the 3′ ends of the PCR products in this step. Thus, if a PCR amplicon is to be cloned into TA vectors, the final extension step of 30 minutes is recommended to ensure proper 3′-dA tailing and efficient PCR cloning (learn more about TA cloning).

1. Breslauer K, Frank R, Blöcker H et al. (1986) Predicting DNA duplex stability from the base sequence. Proc Natl Acad Sci U S A 83(11):3746–37450.
2. Rychlik W1, Spencer WJ, Rhoads RE (1990) Optimization of the annealing temperature for DNA amplification in vitro. Nucleic Acids Res 18(21):6409–6412.
3. Chester N, Marshak DR (1993) Dimethyl sulfoxide-mediated primer Tm reduction: a method for analyzing the role of renaturation temperature in the polymerase chain reaction. Anal Biochem 209(2):284–290.