How does polymerase chain reaction (PCR) work?

How Polymerase Chain Reaction (PCR) Works

PCR is a molecular technique that exponentially amplifies specific DNA sequences through repetitive cycles of three temperature-dependent steps: denaturation (separating DNA strands at ~98°C), annealing (primer binding at ~60°C), and extension (DNA synthesis at ~72°C), producing approximately 100 billion copies from a single DNA molecule within hours. 1, 2

Core Components Required

PCR requires five essential components that work together to enable DNA amplification 1, 2:

• DNA template - The target genetic material to be amplified (can be genomic DNA or cDNA) 1
• Two primers - Short oligonucleotide sequences (typically 18-25 base pairs) that flank and define the target region to be amplified 2
• DNA polymerase - Heat-stable Taq polymerase enzyme that synthesizes new DNA strands 1, 2
• Nucleotides (dNTPs) - The building blocks (dATP, dTTP, dGTP, dCTP) for new DNA synthesis 1
• Buffer solution - Maintains optimal pH and provides necessary cofactors (typically includes MgCl₂) for enzyme activity 1

The Three-Stage Amplification Cycle

Each PCR cycle consists of three distinct temperature-controlled stages that are repeated 24-40 times 3, 1:

Denaturation Phase

• Heat the reaction mixture to 94-98°C for 10 seconds to 3 minutes 3
• This high temperature breaks hydrogen bonds between complementary DNA strands, separating the double helix into single strands 1, 2
• The initial denaturation step is typically longer (3 minutes) to ensure complete strand separation 3

Annealing Phase

• Cool the reaction to 50-65°C (typically ~60°C) for 20 seconds 3
• At this lower temperature, the primers bind (hybridize) to their complementary sequences on the single-stranded DNA template 1, 2
• Primer specificity is critical - primers must be designed to bind only to the target sequence 3

Extension Phase

• Raise temperature to 72°C for 30 seconds to several minutes 3
• Taq polymerase synthesizes new DNA strands by adding nucleotides complementary to the template, starting from the primers 1, 2
• Extension time depends on amplicon length (typically 1 minute per 1000 base pairs) 3
• A final extension at 72°C for 5 minutes ensures completion of all DNA synthesis 3

Exponential Amplification Principle

The power of PCR lies in its exponential amplification capacity 1, 2:

• After each complete cycle, the amount of target DNA doubles 1, 4
• With 25-30 cycles, a single DNA molecule can be amplified to produce millions to billions of copies 1, 2
• This exponential growth follows the formula: 2ⁿ copies (where n = number of cycles) 4
• Typical PCR protocols use 24-29 cycles to minimize amplification bias while achieving sufficient product 3

Critical Technical Considerations

Primer Design and Optimization

• Primers must amplify regions spanning at least 25 bp beyond the target sequence on both sides for accurate analysis 3
• Testing 3-5 primer pairs for each new target site is recommended to ensure specificity and high efficiency 3
• Unoptimized primers can bind nonspecifically throughout the genome, producing multiple unwanted amplification products 3

Cycle Number Optimization

• Excessive PCR cycles introduce significant amplification bias 3
• Use quantitative PCR (qPCR) to determine the minimum cycle number corresponding to the top of the linear amplification range 3
• For deletions ≤50 bp, bias is typically <10%, but for larger deletions, bias can reach 30-40% 3

Quality Control Measures

• Confirm efficient amplification using gel electrophoresis after each PCR step 3
• Include appropriate positive and negative controls in every run 3, 5
• Use high-fidelity DNA polymerases to minimize errors during amplification 3

Reverse Transcription PCR (RT-PCR) for RNA

When the starting material is RNA (not DNA), an additional reverse transcription step precedes standard PCR 3:

• Reverse transcriptase enzyme converts RNA into complementary DNA (cDNA) using a poly-dT primer that binds to the polyadenylated tail of RNA 3
• The resulting cDNA then serves as the template for standard PCR amplification 3
• RT-PCR is widely used for detecting viral RNA (such as SARS-CoV-2) and studying gene expression 3
• One-step RT-PCR (combining reverse transcription and PCR in one reaction) minimizes contamination risk but introduces additional variability 3

Advanced PCR Variants

Beyond conventional PCR, several specialized techniques have been developed 3, 1:

• Quantitative PCR (qPCR) - Uses fluorescent probes to measure DNA quantity in real-time during amplification 3
• Digital droplet PCR (ddPCR) - Partitions samples into thousands of droplets for absolute quantification, particularly useful for low viral loads and detecting rare variants 3
• Triplet repeat-primed PCR (TP-PCR) - Specialized technique for detecting repeat expansions in genetic diseases like myotonic dystrophy 3
• Nested PCR - Uses two sets of primers in successive reactions to increase specificity 1

Common Pitfalls and How to Avoid Them

• Sample contamination - Use separate areas for pre- and post-PCR work; include no-template controls in every run 3
• RNA degradation - Collect samples in RNAlater reagent and verify RNA integrity using housekeeping genes or capillary electrophoresis 3
• Primer dimers - Optimize primer concentrations (typically 0.5 μM final concentration) and annealing temperatures 3
• Incomplete denaturation - Ensure initial denaturation is sufficient (98°C for 3 minutes) 3
• Detection of single bands - A single PCR band does not confirm diagnosis; approximately 25% of individuals are homozygous for normal alleles, requiring confirmatory testing 3