describe and explain the steps involved in the polymerase chain reaction (PCR) to clone and amplify DNA, including the role of Taq polymerase

Published by Patrick Mutisya · 14 days ago

Cambridge A-Level Biology 9700 – Principles of Genetic Technology: PCR

Principles of Genetic Technology – Polymerase Chain Reaction (PCR)

Learning Objective

Describe and explain the steps involved in the polymerase chain reaction (PCR) used to clone and amplify DNA, including the specific role of Taq polymerase.

Overview of PCR

Polymerase Chain Reaction is an in‑vitro technique that produces millions of copies of a specific DNA fragment from a minute starting amount. The reaction proceeds through repeated cycles of three temperature‑dependent steps, each of which is mediated by a thermostable DNA polymerase (most commonly Taq polymerase).

Key Components of a PCR Reaction

ComponentFunctionTypical Concentration
Template DNASource of the target sequence to be amplified0.1–10 ng (for plasmid) or 10–100 ng (for genomic DNA)
Primers (forward & reverse)Short single‑stranded oligonucleotides that define the start and end of the region to be amplified0.1–0.5 µM each
dNTPs (deoxynucleotide triphosphates)Building blocks for new DNA synthesis200 µM each
Taq DNA polymeraseThermostable enzyme that adds nucleotides to the 3′‑end of primers0.5–2.5 U per 50 µL reaction
MgCl2Co‑factor required for polymerase activity; influences fidelity and yield1.5–3.0 mM
Buffer (usually Tris‑KCl)Maintains optimal pH and ionic strengthProvided as 10× concentrate

Step‑by‑Step Cycle of PCR

  1. Denaturation (94–98 °C, 20–30 s)

    Double‑stranded DNA melts into single strands, breaking hydrogen bonds between complementary bases.

  2. Annealing (50–65 °C, 20–40 s)

    Primers bind (anneal) to their complementary sequences on the single‑stranded template. The exact temperature depends on primer length and GC content and is usually calculated using the formula

    \$T_m = 2^\circ\text{C}\times(A+T) + 4^\circ\text{C}\times(G+C)\$

    where A, T, G, and C are the numbers of each base in the primer.

  3. Extension/Elongation (72 °C, 30 s–1 min per kb)

    Taq polymerase synthesises a new DNA strand by adding dNTPs to the 3′‑hydroxyl end of each primer. The enzyme’s optimal activity is at 72 °C, a temperature at which the DNA remains largely single‑stranded.

Complete PCR Protocol

  1. Prepare a master mix containing all reagents except the template DNA.

  2. Aliquot the master mix into individual PCR tubes.

  3. Add the template DNA to each tube.

  4. Place the tubes in a thermal cycler and run the program:

    • Initial denaturation: 94–98 °C for 2–5 min (activates Taq polymerase).

    • 30–35 cycles of:

      1. Denaturation: 94–98 °C for 20–30 s.

      2. Annealing: 50–65 °C for 20–40 s (temperature set according to primer Tm).

      3. Extension: 72 °C for 30 s per kb of target DNA.

    • Final extension: 72 °C for 5–10 min (ensures complete synthesis of all strands).

    • Hold at 4 °C indefinitely until samples are retrieved.

  5. Analyse the amplified product by agarose gel electrophoresis to confirm size and yield.

Role of Taq Polymerase

Taq polymerase, isolated from the thermophilic bacterium Thermus aquaticus, possesses several properties that make it indispensable for PCR:

  • Thermostability: Retains activity after repeated exposure to the high denaturation temperature (94–98 °C), eliminating the need to add fresh enzyme each cycle.

  • Optimal activity at 72 °C: Matches the extension step temperature, allowing rapid synthesis (≈1 kb per minute).

  • 5′→3′ polymerase activity: Extends primers by adding nucleotides to the 3′‑OH end.

  • Lack of 3′→5′ exonuclease (proofreading) activity: Results in a relatively high error rate (\overline{1} error per 9,000 nucleotides), which is acceptable for many applications (e.g., cloning, diagnostics) but may be undesirable for high‑fidelity requirements.

Applications of PCR in Genetic Technology

  • Cloning of specific genes into plasmid vectors.

  • Diagnostic detection of pathogens (e.g., viral load testing).

  • Forensic DNA profiling.

  • Quantitative PCR (qPCR) for measuring gene expression.

  • Site‑directed mutagenesis and generation of recombinant DNA constructs.

Suggested diagram: Schematic of a PCR cycle showing denaturation, primer annealing, and extension by Taq polymerase, with temperature profile plotted over time.

Key Points to Remember

  • Each PCR cycle theoretically doubles the amount of target DNA; after 30 cycles, >109 copies can be generated.

  • Accurate primer design (length 18–25 nt, 40–60 % GC, minimal secondary structures) is critical for specificity.

  • Mg2+ concentration must be optimised; too low reduces yield, too high increases non‑specific amplification.

  • Hot‑start PCR (using a modified Taq or a physical barrier) reduces primer‑dimer formation and improves specificity.