AP Syllabus focus:
‘Polymerase chain reaction amplifies selected DNA fragments through cycles of denaturation, primer annealing, and extension by DNA polymerase.’
Polymerase chain reaction (PCR) is a core biotechnology method used to make many copies of a specific DNA region quickly and selectively. Understanding its reagents, temperature cycling, and sources of error is essential for interpreting PCR-based results.
What PCR does
PCR is an in vitro DNA amplification technique that targets a sequence between two primers and exponentially increases the number of copies through repeated temperature cycles.
Polymerase chain reaction (PCR): A laboratory method that amplifies a specific DNA segment using repeated cycles of denaturation, primer annealing, and DNA synthesis by a DNA polymerase.
PCR is powerful because it can begin with very small amounts of DNA and still generate enough product for downstream analysis.
Essential components (what must be in the tube)
PCR requires a precise mixture; missing or poorly designed components often cause failure or non-specific products.
Template DNA: Contains the target region to be copied (may be genomic DNA, plasmid DNA, or cDNA).
Two primers (forward and reverse): Short single-stranded DNA oligonucleotides that define the start and end of the amplified region.
Thermostable DNA polymerase: An enzyme that survives high temperatures (commonly Taq polymerase) and extends primers.
dNTPs: Deoxyribonucleotide triphosphates (A, T, G, C) used as building blocks.
Buffer + Mg: Maintains optimal pH/ionic conditions; Mg is a critical polymerase cofactor.
Thermocycler: Instrument that rapidly changes temperatures in a programmed sequence.
Primer: A short single-stranded DNA sequence that base-pairs to a complementary template region and provides a free 3′-OH group for DNA polymerase to begin synthesis.
Primers must flank the target on opposite strands and be oriented so synthesis proceeds toward the region to be amplified.
The PCR cycle (three repeating stages)
PCR works by repeating the same three biochemical steps, each controlled by temperature.
This diagram summarizes the core PCR thermocycle: high-temperature denaturation separates strands, lower-temperature annealing allows primers to hybridize, and extension at the polymerase optimum synthesizes new DNA. Seeing the stages side-by-side makes it easier to connect temperature changes to the molecular events that drive amplification. Source
One cycle doubles the number of target molecules (in ideal conditions), so repetition yields exponential amplification.
1) Denaturation
Temperature is raised (often ~94–98°C).
Hydrogen bonds between DNA strands break.
Double-stranded DNA becomes single-stranded templates.
2) Primer annealing
Temperature is lowered (often ~50–65°C).
Primers bind (anneal) to complementary sequences on the single-stranded templates.
Annealing temperature affects specificity:
Too low → primers bind non-specifically, producing extra bands/products.
Too high → primers may not bind efficiently, reducing yield.
3) Extension (elongation)
Temperature is set to the polymerase optimum (often ~72°C for Taq).
DNA polymerase extends from each primer’s 3′ end, adding dNTPs to synthesize new DNA 5′ → 3′.
The polymerase copies the sequence between the primer binding sites, generating double-stranded products.
This figure traces how forward and reverse primers define the ends of the amplicon and how DNA polymerase extends from each primer’s 3′ end in the direction. It also illustrates why “correct-length” products become dominant after the first few cycles, setting up true exponential amplification in later cycles. Source
Why amplification becomes exponential
Early cycles produce products of varying lengths, but after a few cycles the dominant products are the exact fragment bounded by both primers. Because newly made DNA strands become templates in later cycles, the number of correctly sized target fragments increases rapidly.
This amplification plot shows how PCR product accumulation progresses through an early exponential (geometric) phase, then transitions to linear growth and finally a plateau as reagents become limiting. It helps connect the idea of “exponential amplification” to what actually happens across cycles in real reactions and why yields eventually level off. Source
Key ideas that control what gets amplified:
Primer binding sites determine the boundaries of the product.
A primer must match the template well enough at its 3′ end for extension to proceed.
The polymerase must remain active through repeated heating, which is why thermostable enzymes are used.
Good practice: controls and common problems
PCR results are only meaningful when appropriate controls and careful design are used.
Controls
Negative control (no template control): Contains all reagents except template DNA; any product suggests contamination.
Positive control: Uses a template known to amplify; confirms reagents and cycling conditions work.
Common issues to recognise
Contamination: Tiny amounts of stray DNA can be amplified; use clean technique and separated work areas.
Primer-dimers: Primers anneal to each other and get extended, creating small non-target products.
Non-specific amplification: Primers bind unintended sites; improve primer design or raise annealing temperature.
Low yield: Insufficient template, suboptimal Mg, degraded reagents, or annealing temperature too high.
FAQ
Design focuses on specificity and efficiency. Key considerations include:
Similar melting temperatures ($T_m$) for both primers
Minimal self-complementarity to reduce primer-dimers
Avoidance of repetitive sequences
A specific 3′ end to reduce mismatched extension
Hot-start PCR keeps the polymerase inactive until the initial high-temperature step.
This reduces non-specific amplification that can occur when primers bind at room temperature during reaction setup.
Mg$^{2+}$ stabilises primer–template binding and is required for polymerase activity.
Too much Mg$^{2+}$ can increase non-specific products; too little can reduce yield or stop amplification.
RNA must first be converted to complementary DNA (cDNA) using reverse transcriptase.
PCR then amplifies the cDNA, not the RNA, using standard cycling and DNA polymerase.
Amplification slows as:
dNTPs and primers become limiting
polymerase activity decreases over many cycles
product strands re-anneal with each other instead of primers binding
inhibitors in the sample reduce enzyme efficiency
Practice Questions
State the three main stages of a PCR cycle and describe what happens in each stage. (2 marks)
Denaturation: DNA strands separate (1)
Annealing: primers bind to complementary template sequences (1)
Extension: DNA polymerase extends from primers to synthesise new DNA (1)
(Any two correct stages with correct descriptions = 2)
Explain how primers and temperature changes allow PCR to selectively amplify a target DNA fragment from a mixture of DNA. (5 marks)
High temperature denatures double-stranded DNA to single strands (1)
Lower temperature allows primers to anneal by complementary base pairing (1)
Primers flank the target region and define the ends of the fragment amplified (1)
Thermostable DNA polymerase extends from primer 3′ ends to synthesise DNA 5′ to 3′ (1)
Repeated cycles cause newly synthesised DNA to act as template, leading to exponential amplification of the target (1)
