What Is PCR? Polymerase Chain Reaction Explained for Biomedical Students

Polymerase chain reaction (PCR) is a fundamental molecular biology technique that amplifies specific DNA sequences from tiny starting quantities. First developed by Kary Mullis in 1983 (Nobel Prize, 1993), PCR is now indispensable in clinical diagnostics, forensic science, research, and public health. Understanding PCR underpins much of modern biomedical science.

The Three Steps of PCR

Each PCR cycle consists of three steps: (1) Denaturation (94–98°C): the double-stranded DNA template is heated to separate the two strands. (2) Annealing (50–65°C): short synthetic oligonucleotide primers bind (anneal) to their complementary sequences flanking the target region on each template strand. Primer design is critical — primers are typically 18–25 bp long and selected for specific melting temperature and lack of self-complementarity. (3) Extension (72°C): Taq DNA polymerase (isolated from Thermus aquaticus, a thermophilic bacterium) synthesises new DNA from the primer, using the template strand and adding dNTPs (deoxynucleoside triphosphates). After 25–35 cycles, the target region has been amplified exponentially — over one billion copies from a single template molecule.

Key Components of a PCR Reaction

A standard PCR reaction contains: DNA template; forward and reverse primers; Taq DNA polymerase (or a higher-fidelity polymerase such as Phusion or Q5 for applications requiring accuracy); dNTPs (dATP, dCTP, dGTP, dTTP); MgCl₂ (cofactor for polymerase); and a buffer to maintain pH. The magnesium concentration is critical — too little reduces yield; too much causes non-specific amplification.

RT-PCR: Detecting RNA Targets

Reverse transcription PCR (RT-PCR) adds a step before the standard PCR: RNA is first converted to complementary DNA (cDNA) using reverse transcriptase enzyme. This allows amplification of RNA targets — crucial for detecting RNA viruses (e.g. SARS-CoV-2, influenza, HIV), for gene expression analysis (measuring mRNA levels), and for detecting alternatively spliced transcripts. The term RT-PCR is sometimes confused with real-time PCR (see below); one-step RT-PCR kits combine both the reverse transcription and PCR amplification in a single tube.

qPCR: Quantitative Real-Time PCR

Quantitative PCR (qPCR) monitors amplification in real-time using fluorescent reporters — either intercalating dyes (e.g. SYBR Green, which fluoresces when bound to double-stranded DNA) or sequence-specific probes (e.g. TaqMan probes, which are cleaved during extension to release fluorescence). The cycle threshold (Ct or Cq) is the cycle number at which fluorescence crosses a threshold — a lower Ct indicates more starting template. qPCR is used clinically to quantify viral loads (HIV RNA, HBV DNA, CMV), monitor minimal residual disease in haematological malignancies, and assess gene expression.

Clinical Applications of PCR

PCR is used across clinical microbiology and molecular diagnostics: detecting respiratory pathogens (flu, RSV, SARS-CoV-2) in nasopharyngeal swabs; diagnosing sexually transmitted infections (chlamydia, gonorrhoea, trichomonas); detecting MRSA and other resistant organisms; diagnosing genetic conditions via mutation detection; blood donor screening for HIV, HBV, HCV; and detecting BCR-ABL fusion transcript in CML for minimal residual disease monitoring. Multiplex PCR panels can simultaneously detect 15–20+ targets in a single reaction.

References

1. Mullis KB. The unusual origin of the polymerase chain reaction. Sci Am. 1990;262(4):56-65.
2. NIH National Human Genome Research Institute. Polymerase Chain Reaction (PCR). genome.gov
3. Bustin SA, et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009.