The polymerase chain reaction, or PCR, is one of the most well-known techniques in molecular biology. Replication of single-stranded DNA from a template using synthetic primers and a DNA polymerase was first reported as early as the 1970s [1,2]. Nevertheless, the PCR method as we know it today to amplify target DNA was not developed as a research tool until 1983, by Kary Mullis [3,4]. Since then, PCR has become an integral part of molecular biology, with applications ranging from basic research to disease diagnostics, agricultural testing, and forensic investigation. For his invention, Kary Mullis was awarded the Nobel Prize in Chemistry in 1993.

PCR

PCR is a biochemical process capable of amplifying a single DNA molecule into millions of copies in a short time. Amplification is achieved by a series of three steps: (1) denaturation, in which double-stranded DNA templates are heated to separate the strands; (2) annealing, in which short DNA molecules called primers bind to flanking regions of the target DNA; and (3) extension, in which DNA polymerase extends the 3′ end of each primer along the template strands. These steps are repeated (“cycled”) 25–35 times to exponentially produce exact copies of the target DNA (Figure 1).

Over the years, the fundamental principles of PCR have remained the same, but methods have evolved with vast performance improvements to DNA polymerases and reagents, as well as innovations in instrumentation and the plastic vessels that hold the reactions.

Figure 1. Three steps of PCR─denaturation, annealing, and extension─as shown in the first cycle, and the exponential amplification of target DNA with repeated cycling.
 

DNA polymerases

DNA polymerases are critical components in PCR, since they synthesize the new complementary strands from the single-stranded DNA templates. All DNA polymerases possess 5′→ 3′ polymerase activity, which is the incorporation of nucleotides to extend primers at their 3′ ends in the 5’ to 3’ direction (Figure 2).

In the early days of PCR, the Klenow fragment of DNA polymerase I from E. coli was used to generate the new daughter strands [3]. However, this E. coli enzyme is heat-sensitive and easily destroyed at the high denaturing temperatures that precede the annealing and extension steps. Thus, the enzyme needed to be replenished at the annealing step of each cycle throughout the process.

The discovery of thermostable DNA polymerases proved to be an important advancement, opening tremendous opportunities for the improvement of PCR methods by enabling longer-term stability of the reactions. One of the best-known thermostable DNA polymerases is Taq DNA polymerase, isolated from the thermophilic bacterial species Thermus aquaticus in 1976 [5,6]. In the first report in 1988 [7], researchers demonstrated Taq DNA polymerase’s retention of activity above 75°C, making continuous cycling without manual addition of fresh enzyme possible, and thus enabling workflow automation. Furthermore, compared to E. coli DNA polymerase, Taq DNA polymerase produced longer PCR amplicons with higher sensitivity, specificity, and yield. For all the aforementioned reasons, Taq DNA polymerase was named “Molecule of the Year” by the journal Science in 1989 [8].

Although Taq DNA polymerase significantly improved PCR protocols, the enzyme still presented some drawbacks. Taq DNA polymerase is relatively unstable above 90°C during denaturation of DNA strands. This is especially problematic for DNA templates with high GC content and/or strong secondary structures that require higher temperatures for separation. The enzyme also lacks proofreading activity; therefore, Taq DNA polymerase can misincorporate nucleotides during amplification. Where sequence accuracy is critical, PCR amplicons with errors are not desirable for cloning and sequencing. In addition, the error-prone nature of Taq DNA polymerase contributes to its inability to amplify fragments longer than 5 kb in general. To overcome such shortcomings, better-performing DNA polymerases are continually being developed to harness the power of PCR across a variety of biological applications (learn more about DNA polymerase characteristics).

Figure 2. DNA polymerase extending the 3′ end of a PCR primer in the 5′ to 3′ direction.

Thermal cyclers

A thermal cycler is an instrument that automates temperature cycling and incubation times for PCR. Prior to the introduction of thermal cyclers, PCR was a laborious process involving the transfer of samples between water baths of different temperatures, and requiring precise timing of each step. The thermal cycler, together with the discovery of Taq DNA polymerase, made automation of PCR a reality. The first automated thermal cycler for PCR was introduced to the market by PerkinElmer and Cetus as a joint venture in 1985 [9]. Since then, improvements have been made in the utility, design, temperature control, and cycling speed of thermal cyclers (Figure 3). Thermal cyclers also paved the way for the development of quantitative PCR instruments that combine PCR amplification with real-time detection of PCR product accumulation (learn more about quantitative PCR).

Figure 3. Animated depiction of evolution of thermal cyclers over the years.

References

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