Polymerase Chain Reaction (PCR):  Steps, Types, Applications

In December 2019, a cluster of patients in Wuhan, China, developed severe pneumonia of unknown cause. Within two weeks, a novel coronavirus had been identified, its genome sequenced, and a PCR diagnostic test designed. By January 2020, laboratories across the world were running that test. The speed of that response—from unknown pathogen to diagnostic tool in under a month—was possible because of one technique: polymerase chain reaction.

PCR does not just detect pathogens. It amplifies a specific DNA or RNA sequence from a complex biological sample (blood, sputum, CSF, urine, tissue) until enough copies exist to be detected and characterised. A single copy of a viral genome in a milliliter of blood, invisible to culture and undetectable by serology, becomes a confirmed positive after PCR amplification. This is why PCR has become the diagnostic gold standard for tuberculosis, HIV, hepatitis C, COVID-19, and dozens of other infections.

Figure: I was too excited when our lab got the Real-Time PCR first time

Polymerase chain reaction (PCR) is an in vitro molecular technique that amplifies a specific target DNA or RNA sequence exponentially, producing up to 10⁷ copies from a single starting template within a few hours. It was developed in 1983 by American biochemist Kary Mullis, who was awarded the Nobel Prize in Chemistry in 1993 for this discovery.

Figure: PCR Cycle and Target copies

Why PCR Changed Diagnostic Microbiology

Before PCR, detecting a pathogen required either growing it in culture (which takes days and fails for fastidious or non-cultivable organisms) or detecting the host's antibody response (which takes weeks to develop and cannot distinguish current from past infection). Both approaches have fundamental limitations that PCR bypasses.

PCR directly detects the pathogen's nucleic acid (its DNA or RNA) regardless of whether the organism is alive, dead, cultivable, or present in tiny quantities. This gives PCR four critical advantages in clinical microbiology:

Speed: Results in hours rather than days. Culture of Mycobacterium tuberculosis takes 6–8 weeks; PCR on sputum can confirm TB in a single working day.

Sensitivity: Can detect as few as 1–10 copies of target DNA per reaction. Culture may miss organisms present in low numbers; PCR does not.

Specificity: Primers are designed to bind only the target sequence. A positive result identifies the specific organism or gene — not just "something grew."

Versatility: Works on organisms that cannot be cultured (viruses, some parasites), on degraded or archival samples (formalin-fixed tissue, dried blood spots), and on specimens with mixed flora where culture is uninterpretable.

Components of PCR

Every PCR reaction requires five essential components:

Primer: A short, synthetic, single-stranded DNA sequence complementary to one end of the target region. Two primers are used: the forward primer binds upstream of the target; the reverse primer binds downstream. Together they define the exact sequence to be amplified.

Taq polymerase: Named after Thermus aquaticus, a thermophilic bacterium isolated from hot springs in Yellowstone National Park. Its heat stability (remaining active at 72°C and surviving 94°C) is what makes automated PCR cycling possible.

Steps of PCR

PCR is performed in a thermocycler; an instrument that raises and lowers temperature in programmed cycles automatically. Each cycle consists of three steps:

Figure: Steps of PCR (denaturation, annealing, extension)

Step 1: Denaturation (94–96°C)

The double-stranded DNA template is heated to 94–96°C. At this temperature, the hydrogen bonds between complementary base pairs break, separating the two strands into single-stranded templates. This makes each strand accessible for primer binding in the next step.

Step 2: Annealing (45–65°C)

The temperature is lowered to 45–65°C (the exact temperature depends on the primer design — typically 5°C below the primer melting temperature). The primers bind to their complementary sequences on each single-stranded template. The forward primer binds to one strand; the reverse primer binds to the other, flanking the target region.

Step 3: Extension (72°C)

The temperature is raised to 72°C — the optimal temperature for Taq polymerase activity. Starting from each primer, Taq polymerase reads the template strand and synthesizes a new complementary DNA strand by incorporating dNTPs. Extension proceeds in the 5' to 3' direction at approximately 1,000 bases per minute.

After the first cycle, two double-stranded DNA copies exist where one existed before. After 30 cycles, the target sequence is amplified by a factor of 2³⁰ = approximately 10⁹ copies — though practically, amplification efficiency is never 100%, and yields of 10⁶–10⁷ copies are typical after 30–40 cycles.

Detection of PCR Products

Amplified products (amplicons) are detected by one of two methods:

Gel electrophoresis: The PCR product is run on an agarose gel stained with ethidium bromide. A band at the expected size confirms amplification of the correct target. Simple, inexpensive, but qualitative only — cannot quantify the original amount of target.

Labelled probe detection: A probe complementary to an internal sequence of the amplicon hybridizes to the product and generates a signal (fluorescent, colorimetric, or chemiluminescent). Probe-based detection adds specificity — confirming the amplicon is the correct target, not a non-specific product. This is the basis of real-time PCR.

Types of PCR Used in Clinical Microbiology

Several modifications of standard PCR have been developed for specific diagnostic needs. Understanding which type to use and why, is more useful than memorizing definitions.

Real-time PCR (Quantitative PCR, qPCR)

Amplification and detection occur simultaneously in a closed tube using fluorescent probes or dyes. The instrument measures fluorescence after each cycle, generating a real-time amplification curve. This enables quantitation — calculating the exact number of target copies in the original sample, not just detecting presence or absence.

Clinical use: HIV viral load, HCV viral load, HBV viral load, COVID-19 PCR, TB quantification, CMV monitoring in transplant patients.

Key advantage: No post-PCR handling required — closed tube eliminates carry-over contamination. Faster and more quantitative than conventional PCR.

See full article: Real-time PCR: Principles and Applications

Reverse Transcriptase PCR (RT-PCR)

Standard PCR amplifies DNA. RT-PCR adds a reverse transcription step before amplification — the enzyme reverse transcriptase converts the RNA template into complementary DNA (cDNA), which is then amplified by standard PCR.

Clinical use: Detection of RNA viruses; HIV, hepatitis C, dengue, SARS-CoV-2, influenza, enteroviruses. Also used to detect bacterial and parasitic rRNA (indicating viable organisms) and to study mRNA gene expression.

Key advantage: The only PCR method that can amplify RNA targets directly.

See full article: Reverse Transcriptase PCR: Principles and Applications

Nested PCR

Two successive PCR reactions use two sets of primers. The first reaction (outer primers) amplifies a large fragment; the second reaction (inner or nested primers) amplifies a smaller region within that fragment. The double primer system dramatically increases both sensitivity and specificity.

Clinical use: Detection of organisms present in very low quantities — Rickettsia, Bartonella, M. tuberculosis in paucibacillary samples, herpesvirus and enterovirus in CSF, Leishmania in tissue.

Key advantage: Higher sensitivity than single-round PCR for samples with very low pathogen load.

See full article: Nested PCR: Principle and Applications

Multiplex PCR

Multiple primer pairs targeting different sequences are included in a single PCR reaction, allowing simultaneous amplification of several targets at once.

Clinical use: Detection of multiple meningitis pathogens (S. pneumoniae, H. influenzae, N. meningitidis) from a single CSF sample; respiratory panel testing; BioFire FilmArray syndromic panels.

Key advantage: Multiple pathogens detected from one reaction, reducing cost, time, and sample volume.

See full article: Multiplex PCR: Principle, Applications, and Limitations

Other PCR Types

Several additional PCR modifications are used in research and specialized diagnostic settings:

RT-PCR vs. Real-time PCR: The Most Common Confusion

These two terms are routinely confused including in clinical settings during the COVID-19 pandemic, when "RT-PCR" was used by media and health authorities to mean different things simultaneously.

The practical rule: RT-PCR refers to the template type (RNA). Real-time PCR refers to the detection method (fluorescence during cycling). A test can be both — and most modern viral load assays are.

How to Remember

PCR = molecular photocopier with a temperature dial. A photocopier takes one document and makes many copies. PCR takes one DNA sequence and makes millions. The thermocycler is the machine; the three temperatures are the three steps: high heat separates (94°C), cool down for primers to bind (50–65°C), warm up for extension (72°C).

"Denature, Anneal, Extend" — DAE, like the name. The three steps in order: Denaturation (94°C) → Annealing (50–65°C) → Extension (72°C). DAE. Each step has one job: separate, bind, build.

Taq polymerase = hot spring survivor. Thermus aquaticus lives in boiling hot springs. Its polymerase survives 94°C without denaturing — the property that makes automated PCR cycling possible. Without a heat-stable polymerase, fresh enzyme would have to be added after every denaturation step.

PCR type selection: the three clinical questions:

1. Is the target RNA? → Need RT-PCR
2. Do I need a number (viral load), not just yes/no? → Need real-time (qPCR)
3. Is the target present in tiny amounts or is sensitivity critical? → Consider nested PCR
4. Are multiple pathogens possible from one sample? → Consider multiplex PCR

Key exam facts in one table

References

1. Mullis, K. B. (1990). The unusual origin of the polymerase chain reaction. Scientific American, 262(4), 56–65.
2. Murray, P. R., Rosenthal, K. S., & Pfaller, M. A. (2021). Medical Microbiology (9th ed.). Elsevier.
3. Mahon, C. R., Lehman, D. C., & Manuselis, G. (2018). Textbook of Diagnostic Microbiology (6th ed.). Elsevier.
4. Forbes, B. A., Sahm, D. F., & Weissfeld, A. S. (2007). Bailey & Scott's Diagnostic Microbiology (12th ed.). Mosby Elsevier.
5. Chang-Hui Shen. (2019). Amplification of Nucleic Acids. Diagnostic Molecular Biology. Academic Press. https://doi.org/10.1016/B978-0-12-802823-0.00009-2
6. Clinical and Laboratory Standards Institute (CLSI). (2016). Clinical Microbiology Procedures Handbook (4th ed.). American Society of Microbiology.