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Polymerase Chain Reaction (PCR) is a molecular technology developed by Nobel laureate Kary Mullis in the 1980s that allows the fast and inexpensive amplification of DNA fragments in vitro (1). It has since become a fundamental tool in genetic and molecular research as large amounts of the target DNA are often required for DNA experimentation (2)(3). Some applications that rely on PCR technology include DNA sequencing (e.g. Human Genome Project), DNA fingerprinting, forensics, detection of bacteria or viruses (particularly AIDS) and diagnosis of hereditary disease (4)(5). Due to its ability to generate large quantities of DNA from a small amount of nucleic acid, PCR is a very efficient way to amplify DNA and thus is sometimes referred to as “molecular photocopying” (2)(3)(6).

In a nutshell, PCR is essentially a series of 20-40 repeated cycles of heating and cooling (called thermal cycling) that facilitate DNA replication through enzymatic reactions. The amount of target sequence doubles with each cycle which leads to an exponential amplification represented by 2^{(# of cycles)}. Therefore, a PCR thermal cycling of 40 repeats will generate 1,099,511,627,776 copies of DNA from a single copy of the template. The function and purpose of each step in a PCR Reaction are discussed below (Figure 1):

Initialization

The reaction is heated to 94–96°C (or 98°C if extremely thermostable DNA polymerases are used), for 2-10 minutes. This step activates the DNA polymerase and reagents in the reaction as well as denatures other contaminants (if any) in the mixture. In applications such as colony screening, small amounts of cells can be directly used as a template. This initialization step will lyse the cells to release the DNA and denature other cellular proteins including DNases. If a chemical or antibody (such as BlasTaq™ HotStart DNA polymerase) is used during PCR, this step is mandatory to heat activate the enzyme.

Learn more about BlasTaq™ HotStart DNA polymerase (Cat. No. G595) here.

Denaturation

The reaction is heated to 94–98°C for 20–30 seconds. This is the first step of thermal cycling. Double-stranded DNA is denatured in this step as hydrogen bonds between the strands are broken due to the high temperature, a process also known as DNA melting.

Annealing

Primers are oligonucleotides that can bind to specific sequences of the DNA template to guide DNA polymerase replication. To allow primers to anneal to the single-stranded DNA template, the reaction temperature is typically lowered to 50-65°C for 20-40 seconds. The optimal temperature for annealing depends on the melting temperature (T_m) of the primers: the temperature at which half of the DNA duplex dissociates to become single stranded. The primer cannot anneal to the template if the annealing temperature is set too high, and non-specific priming (which leads to non-specific amplification) may happen if the annealing temperature is too low. Thus, the ideal annealing temperature is usually about 3-5°C below the T_m of the primers, high enough for specific primer annealing, but low enough to allow for efficient priming. The primer concentration in the reaction mixture is usually much higher than that of the DNA template so that primer-template hybridization is greatly favored over re-annealing of the template strands. As soon as the primer anneals to the template, the DNA polymerase can start incorporating dNTPs onto the template.

Jump further down the article to learn more about primer design here.

Extension/Elongation

In this step, DNA polymerase binds to the primer/template complex and starts incorporating dNTPs in a 5’ to 3’ direction on the synthesizing strand. This yields a newly synthesized DNA strand complementary to the template strand. The optimal temperature for extension/elongation varies for different DNA polymerases. BlasTaq™ DNA Polymerase (which is heat resistant) works ideally at 72-78°C. The duration of extension depends both on the length of the original template (a.k.a. amplicon) and the speed of the DNA polymerase. Typical DNA polymerases polymerize at a speed of 1-1.5kb/min when it's within its optimal temperature range.

Final Elongation

The reaction mixture is kept at 72-78°C (the optimal working temperature for most polymerases) for 5-15 minutes in the last cycle of elongation. This step ensures that any remaining single-stranded DNA is fully extended after the last PCR cycle.

Final Hold

The temperature is lowered to 4-15°C for an indefinite amount of time for short-term storage of the reaction mixture (6).

To check whether the correct target DNA fragment has been amplified, gel electrophoresis is a quick method to examine the molecular sizes (in bp) of the amplified products. The size(s) of the amplified PCR products are estimated by comparing it to a DNA ladder, a molecular weight marker which contains DNA fragments of known sizes.

Many factors can interfere with a PCR reaction. Some are easy to optimize (e.g. thermal cycling conditions and Mg²⁺ concentration) while others are trickier to manipulate and would require accumulated experience to tackle (e.g. primer design and PCR buffer components/additives). In general, factors that are important in PCR include DNA templates, DNA polymerase, primer design, buffer components, additives and inhibitors, and thermal cycling conditions (6).

The type of DNA template used for the amplification of a target region can affect the PCR efficiency and accuracy. Below, three types of common PCR templates are introduced along with strategies for improving PCR results.

GC Rich Template

GC content refers to the percentage of guanine and cytosine bases of a DNA region. GC rich templates (GC content > 60%) have increased hydrogen bond strength as three hydrogen bonds form between a GC base pair compared to two between an AT base pair. High GC content leads to an increased likelihood of secondary structure generation (7)(8), which are structures formed when complementary regions of a single stranded DNA molecule loop back and form hydrogen bonds (9). Both increased hydrogen bond strength and secondary structures hinder template denaturation and primer annealing in PCR. Secondary structures may also lead to premature termination as a result of polymerase arrest (8). Annealing of primers at alternative binding sites on the template, a.k.a competitive binding is often strong in GC-rich templates which cause incorrect amplicons to be created (7). Promoter regions of most housekeeping genes, tissue-specific genes and tumor suppressor genes are often rich in GC content, posing difficulties for amplification of these templates.

Strategies to improve the amplification of high GC content templates include optimization of thermal cycling conditions and the use of additives. High annealing temperature can help prevent non-specific binding of primers and amplification. The duration of the annealing time must be optimized to be long enough to allow for the primer to anneal properly to the template, but short enough to prevent competitive binding (7). Additives such as DMSO and Betaine can act as secondary structure destabilizers, thus lowering the melting temperature and the denaturation duration and promoting specific primer annealing (10).

AT Rich Templates

AT rich templates often require a lower annealing temperature as the hydrogen bonds between the base pairs are not as strong as in GC rich templates. On the other hand, non-specific primer annealing can occur under low annealing temperatures.

The use of additives such as TMAC can increase the T_m, thus increasing primer specificity. The concentration of TMAC added must be optimized as enzyme amplification can be inhibited at high concentrations of this additive. A lower extension temperature such as 65-68°C for AT rich templates also helps with amplification, as DNA melting at the typical extension temperature of 72°C prevents successful extension of extremely AT rich regions (11).

Long Templates

It is difficult to amplify long templates in PCR while maintaining high yield, specificity and fidelity. Longer templates usually have a higher likelihood of breaks in the DNA template or degradation caused by sample preparation or depurination. Depurination refers to a chemical reaction during which the β-N-glycosidic bond in the purine nucleosides (e.g. A and G) is cleaved to release a nucleic base due to hydrolysis. As Taq DNA polymerase will not extend through apurinic positions during replication, depurination is an important limiting factor of long templates amplification (33). DNA polymerases sometimes mis-incorporate nucleotides onto the 3’ end during replication, which may result in premature termination and the accumulation of truncated products. Thus, the amplification efficiency decreases as the amplicon size increases due to the accumulation of truncated products. Several strategies can be employed to avoid this:

• Using higher pH: Lower pH promotes depurination of templates. Increasing the pH of a PCR reaction protects templates from depurination damage, thus decreasing the number of truncated products and increasing the yield.
• Adding glycerol or DMSO: The processing efficiency of the DNA polymerase becomes important when working with longer templates. DMSO and glycerol are dissociating additives that destabilize DNA and subsequently alter the melting characteristics of the double stranded DNA. In this way, the denaturation and annealing temperatures required are lowered, allowing for faster and more efficient denaturation and annealing steps.
• Increasing extension time: In long PCR templates, the extension time can be increased to allow enough time for the DNA polymerase to add nucldotides. This also decreases the number of truncated products synthesized thus increasing the yield of longer PCR products.
• Using a “proofreading” DNA polymerase: A secondary thermostable DNA polymerase that possesses a 3'-to 5'-exonuclease, or "proofreading," activity can remove misincorporated nucleotides, which greatly improves the ability of the main DNA polymerase to synthesize longer products with higher yield (12)(13).

For a more in-depth discussion of DNA polymerase and its variations please read our PCR – DNA Polymerase Variation knowledge base article.

Primer design is essential for successful PCR. Below are important considerations for a good primer design which ensures specific annealing and facilitates efficient amplicon extension (34)(35)(36)(37).

• The optimal primer length is generally between 18-30 nucleotides. Longer primers promote specific annealing but also have higher melting temperature, while shorter primers bind more easily to the template with lower specificity.
• The optimal melting temperature of primers is between 50°C – 65°C. The forward and reverse primer pairs should have T_m within 5°C of each other, so both primers bind simultaneously and efficiently.
• The annealing temperature (T_a) should be set to be no more than 5°C lower than the T_m which is high enough to promote specific binding and low enough to allow efficient annealing.
• Design primers with GC content between 40%-60% to promote specific annealing. The GC content of the target sequence greatly affects the melting temperature, as sequences with higher GC content have a higher melting temperature.
• Ending the primer (at the 3’ terminal) with Gs or Cs can promote specific target binding. However, there should be no more than 3 Gs or Cs in the last five bases.
• Targeting sequences containing secondary structures may result in poor primer annealing and low yield, and should be avoided if possible.
• Avoid having 4 or more nucleotide, or dinucleotide repeats (e.g. ATATATAT).
• Avoid having complementary sequences within the primer or between the forward and reverse primers which usually lead to self-dimers or primer-dimers. As the primer concentration is much larger than the template concentration in a reaction, the formation of self-dimers and primer-dimers will seriously compete with template-primer annealing and affects product yield.

Some powerful primer design tools available online are include PrimerBLAST and OligoCalc, which can be used to simplify the primer design process.

abm offers Custom Primer Design and Synthesis Services. Read more about the services here

The most common components of PCR buffers are Tris-HCl, Magnesium ions, KCl or other salt solutions, and dNTPs. Co-solvents or additives may also be included to enhance the amplification. Buffer components greatly affect PCR result as they change the pH, cofactors levels, salt concentration and ionic strength.

Thermostable DNA polymerases require a relative stable pH to perform with optimized activity and fidelity. Tris-based buffers are generally used due to their ability to stabilize pH over a broad range of temperatures and changes in reaction mix compositions.

Magnesium is a co-factor for thermostable DNA polymerases and its presence in the reaction is essential for PCR success. For example, the activity of Taq polymerase (the mostly commonly used polymerase) drops dramatically when there is insufficient amount of free Mg²⁺, while its fidelity and amplification specificity is reduced when there is an excess amount of Mg²⁺. The performance of Pfu DNA polymerase, on the other hand, is less dependent on magnesium concentration. Because of this, DNA polymerases are supplied in a Magnesium-free reaction buffer along with a separate aliquot of Mg ion solution to enable researchers to optimize each reaction using varying Mg²⁺ concentration.

Salt can neutralize the negative charges on the phosphate backbone of DNA, thus reducing the repulsion between two DNA strands. Some salt in PCR buffers can stabilize primer-template binding during annealing to help polymerases start extension. A small level of KCl in PCR buffers can greatly increase DNA polymerase activity and the yield of short length products (39), while longer templates are more efficiently amplified at lower concentrations of K⁺. The use of a combination of KCl and (NH₄)₂SO₄ can destabilize hydrogen bonds between mismatched bases, thus promoting specificity of primer annealing. dNTP (deoxynucleoside triphosphate) are building blocks that the DNA polymerases uses to incorporate into the synthesis strand. A balanced mix of dATP, dGTP, dCTP and dTTP is necessary for the best incorporation, although an unbalanced dNTP concentration can sometimes be used to promote DNA polymerases misincorporation in certain applications such as random mutagenesis studies (40).

PCR buffers usually contain various additives to enhance amplification. More information can be found about additives and inibitors in the next section.

To increase the yield, specificity, and consistency of PCR reactions, PCR enhancing agents or additives can be added. Common additives include DMSO, Betaine, Formamide, TMAC, BSA, non-ionic detergents, glycerol, and etc. They are especially important in enhancing PCR amplification of long or GC rich templates, and some of them have been discussed in previous sections. The use of additives needs to be optimized based on the experimental conditions and application, and often require repeated trial and error.

Table 1: Examples of PCR additives and their mechanism of action (3)(8)(14)(15)(16)(17):

PCR is also affected by inhibiting substances present in samples. Some residual inhibitors are present in specific types of DNA samples and will require sample-specific nucleic acid isolation protocols prior to PCR. PCR inhibitors can also be introduced during sample preparation. Inhibitors may degrade or modify the DNA template, disturb annealing of the primers to the DNA template (due to competitive binding of the inhibitor to the template), and degrade, inhibit or alter DNA polymerase activity. Refer to Table 2 for examples of PCR inhibitors and their mechanism of action (18).

Table 2 - Examples of PCR inhibitors and their mechanisms of action

Source: Adapted from PCR inhibitors – occurrence, properties and removal. Schrader, C., Schielke, A., Ellerbroek, L. and Johne, R. s.l. : Journal of Applied Microbiology, July 24th, 2012, Vol 113.

Each of our PCR MasterMixes are tailored to their specific DNA polymerase for the optimal results. See our comprehensive collection of different DNA Polymerases here.

The lengths and temperatures for the thermal cycle in PCR can be modified to promote amplification under different experimental conditions. In some cases, the denaturation cycle can be shortened or a lower temperature used to reduce the chances of nucleotides depurination, which can lead to truncated products or mutations in the PCR products.

Annealing temperature and extension times are the most commonly altered parameters to reduce non-specific amplification and primer-dimer formation. The optimal annealing temperature (T_a) is often within 5°C of the T_m of the primers. Higher T_a leads to increased annealing stringency and specificity, while lower T_a results in more efficient annealing and higher yields. The extension time should be optimized based on the size of the PCR product and the DNA polymerase being used. In general, it takes a minimum of 1 minute per kb of amplicon for non-proofreading DNA polymerases and 2 minutes per kb of amplicon for proofreading DNA polymerases. Excessively long extension times allow the intrinsic 5′→3′ exonuclease activity of Taq DNA polymerase to generate undesired products, so should be avoided.

Typically 25–35 thermal cycles in PCR can synthesize enough products for downstream application. As the number of cycles increase, the risk of undesirable PCR products appearing in the reaction increases, so it is important to set an appropriate number of thermal cycles in PCR (40).

Touchdown PCR is one method of PCR with specially modified thermal cycling conditions to enhance PCR results. In the initial cycles of touchdown PCR, the T_a (annealing temperature) is set higher (usually several degrees above the estimated T_m of primers) to tolerate only specific primer binding to the template and promote amplification of the correct target sequence. T_a is then gradually decreased for every subsequent cycle (e.g. 1-2°C/every second cycle), to allow more efficient primer-binding and amplification. Since the desired products were already amplified during the earlier cycles, they will out-compete the non-specific sequences the primer may risk bidnig to at lower T_a. Touchdown PCR therefore offers a simple and rapid method to optimize PCR with enhanced specificity, efficiency and yield, and is particularly useful for the amplification of difficult templates (41).