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

Principles of Genetic Technology – Polymerase Chain Reaction (PCR)

Learning Objective (AO1)

Describe the principle and each step of the polymerase chain reaction (PCR) used to clone and amplify DNA, and explain the specific role of the thermostable enzyme Taq polymerase.

Context within Genetic Technology (AO1 & AO2)

• PCR is a core recombinant‑DNA technique that works together with restriction enzymes, DNA ligase, cloning vectors, transformation and newer genome‑editing tools (e.g., CRISPR‑Cas9).
• Typical workflow:

  template DNA → PCR amplification → restriction‑enzyme digestion → ligation into a plasmid vector → transformation of a host cell → expression or further modification.

• Understanding PCR underpins the whole “Genetic Technology” topic (Syllabus Topic 19, Cambridge International AS & A Level Biology 9700).

Key Terminology (required by the syllabus)

• Thermostable DNA polymerase – enzyme that remains active after repeated heating cycles (e.g., Taq polymerase).
• Proof‑reading polymerase – DNA polymerase with 3′→5′ exonuclease activity, giving a lower error rate (e.g., Pfu, Phusion).
• Hot‑start PCR – technique that prevents polymerase activity until the initial denaturation step, reducing non‑specific amplification.
• Primer‑dimer – a short, non‑target amplicon formed when primers anneal to each other.
• Annealing temperature (T_m) – the temperature at which 50 % of the primer‑template duplexes are formed.
• Reverse‑transcription PCR (RT‑PCR) – PCR performed on cDNA that has been synthesised from RNA, allowing analysis of gene expression.

Components of a Standard PCR Reaction (AO2)

Step‑by‑Step Cycle of PCR (AO1)

1. Initial Denaturation (94–98 °C, 2–5 min)

   • Fully separates the double‑stranded template.
   • Activates “hot‑start” Taq polymerases that are chemically or antibody‑blocked at lower temperatures.

2. Denaturation (94–98 °C, 20–30 s)

   Melts the DNA strands each cycle, creating single‑stranded templates for primer binding.

3. Anealing (50–65 °C, 20–40 s)

   Primers hybridise to their complementary sequences. The temperature is set 3–5 °C below the calculated T_m of the primers.

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

   • Taq polymerase adds dNTPs to the 3′‑OH end of each primer, synthesising a new strand in the 5′→3′ direction.
   • At 72 °C the enzyme works at its optimum rate (≈1 kb per minute).

One complete set of the three steps (denaturation‑annealing‑extension) constitutes a PCR cycle. After 30–35 cycles the amount of target DNA is theoretically amplified by a factor of 2ⁿ (n = number of cycles), giving >10⁹ copies.

Worked Example – T_m Calculation

Primer sequence: 5′‑ATG CCT GGA TCC GAA TGC‑3′ (20 nt)

• Count bases: A = 4, T = 4, G = 6, C = 6
• Apply the Wallace rule:
  

  T_m = 2 °C × (A + T) + 4 °C × (G + C)
  

  T_m = 2 × (4 + 4) + 4 × (6 + 6) = 16 + 48 = 64 °C

• Set annealing temperature ≈ 64 °C − 3 °C = 61 °C.

Worked Example – Theoretical Yield After 30 Cycles

Assume a single starting molecule (1 copy). After each cycle the copy number doubles.

Copies after n cycles = 2ⁿ
For n = 30: 2³⁰ = 1 073 741 824 ≈ 1 × 10⁹ copies

If the initial template is 10 ng of a 3 kb plasmid (≈3 × 10⁶ bp, 1 pg ≈ 9.1 × 10⁵ bp), the starting copy number is roughly 10 ng ÷ (3 kb × 660 Da) ≈ 5 × 10⁶ copies. After 30 cycles the theoretical yield would be ≈ 5 × 10⁶ × 2³⁰ ≈ 5 × 10¹⁵ copies (≈ 3 µg of product, assuming 100 % efficiency).

Complete PCR Protocol (AO2 – Practical Skills)

1. Prepare a master mix containing buffer, MgCl₂, dNTPs, primers, and Taq polymerase. Make enough for all reactions plus a 10 % excess.
2. Aliquot the master mix into labelled PCR tubes or a 96‑well plate (usually 45 µL per tube).
3. Add the appropriate volume of template DNA (5 µL) to each tube. Include the following controls:

   • Positive control – a template known to amplify with the chosen primers.
   • Negative control (no‑template control, NTC) – contains all reagents except template DNA; detects reagent contamination.
   • Primer‑dimer control (optional) – contains primers but no template to assess primer‑dimer formation.

4. Seal the tubes (optical caps or mineral oil) and place them in a calibrated thermal cycler.
5. Run the temperature programme (example for a 1 kb target):

   Initial denaturation: 95 °C, 3 min
   30 cycles:
   Denaturation      95 °C, 30 s
   Annealing         61 °C, 30 s   (example Tm‑based)
   Extension         72 °C, 1 min
   Final extension:    72 °C, 5 min
   Hold:                4 °C
   

6. After cycling, store the products at –20 °C or proceed directly to analysis.

Role of Taq Polymerase (AO1 & AO2)

• Thermostability – isolated from Thermus aquaticus; retains >90 % activity after repeated 95 °C denaturation steps.
• Optimal activity at 72 °C – matches the extension step, allowing rapid synthesis (~1 kb min⁻¹).
• 5′→3′ DNA synthesis – adds nucleotides to the 3′‑OH of the primer.
• Lacks 3′→5′ exonuclease (proof‑reading) activity – error rate ≈1 error per 9 000 nt (≈0.011 %).

Comparison of Common DNA Polymerases

Hot‑Start PCR & Primer‑Dimer Prevention (AO1)

• Hot‑start enzymes are chemically modified or bound by antibodies that inactivate the polymerase at low temperature. The modification is removed only during the initial denaturation step, reducing non‑specific priming.
• Primer‑dimer control – run a reaction with primers only; a faint ~30–50 bp band indicates excessive primer complementarity. Remedy: lower primer concentration, redesign primers, or use hot‑start Taq.

Reverse‑Transcription PCR (RT‑PCR) – Box

RT‑PCR links PCR to gene‑expression analysis.

1. RNA extraction – isolate total RNA from cells or tissue; treat with DNase to remove contaminating DNA.
2. Reverse transcription – use reverse transcriptase (often with an oligo‑dT or random hexamer primer) to synthesise complementary DNA (cDNA) from the RNA template.
3. PCR amplification – the cDNA serves as the template in a standard PCR using gene‑specific primers.
4. Analysis – agarose gel electrophoresis for presence/size, or quantitative real‑time PCR (qPCR) for expression levels.

Because RNA is single‑stranded and unstable, the reverse‑transcription step is essential; without it, the polymerase cannot amplify the target.

Beyond PCR – Sidebar

• Digital PCR (dPCR) – partitions a sample into thousands of nanoliter reactions; absolute quantification is obtained by counting positive partitions. Provides higher precision than qPCR, especially for low‑abundance targets.
• Loop‑mediated isothermal amplification (LAMP) – amplifies DNA at a constant temperature (60‑65 °C) using a set of 4‑6 primers and a strand‑displacing polymerase. Useful for point‑of‑care diagnostics where a thermocycler is unavailable.
• Both techniques illustrate that while PCR remains the benchmark for versatility and reliability, newer methods can complement or replace it in specific applications.

Designing a Simple PCR‑Based Experiment (AO3)

1. Define the objective – e.g., amplify a 750 bp fragment of the lacZ gene from E. coli genomic DNA for cloning into pUC19.
2. Select primers

   • Length 18–25 nt, 40–60 % GC, T_m ≈ 58–62 °C.
   • Add 5′ restriction sites (EcoRI, HindIII) plus 4–6 extra bases to ensure efficient digestion.

3. Choose controls – positive control (known lacZ template), NTC, and a “no‑polymerase” control to check for contaminating nucleases.
4. Optimise reaction conditions

   • MgCl₂ titration (1.5, 2.0, 2.5 mM).
   • Anealing temperature gradient (55–65 °C).
   • Cycle number (25–35) to avoid plateau effects.

5. Run the PCR and analyse – see “Interpreting PCR Results” below.

Interpreting PCR Results (AO3)

1. Agarose Gel Electrophoresis

• Prepare a 1 % agarose gel (0.5 × TAE or TBE) with a DNA‑binding dye (e.g., SYBR Safe).
• Load 5 µL of each PCR product mixed with loading dye; include a 100 bp DNA ladder.
• Run at 100 V for 30–45 min, then visualise under UV/blue light.
• Band identification

  • Correct size – matches the expected fragment length (e.g., 750 bp).
  • Extra bands – indicate non‑specific amplification; raise annealing temperature or use hot‑start polymerase.
  • Primer‑dimer band (~30–50 bp) – reduce primer concentration or redesign primers.
  • No band in NTC – good; band in NTC – contamination, repeat with fresh reagents.

2. Quantitative PCR (qPCR) – Interpreting Amplification Curves

• Fluorescent dye (SYBR Green) or probe (TaqMan) reports the amount of double‑stranded DNA after each cycle.
• C_t (threshold cycle) – the cycle at which fluorescence exceeds the background.
• ΔΔC_t method (exercise)

  Sample data (triplicate):
  Gene of interest (GOI)   Cₜ   Reference (GAPDH)   Cₜ
  Control                  22.5   18.0               18.0
  Treated                  20.0   17.5               17.5
  Step 1: ΔCₜ = Cₜ(GOI) – Cₜ(Reference)
  Control ΔCₜ = 22.5 – 18.0 = 4.5
  Treated ΔCₜ = 20.0 – 17.5 = 2.5
  Step 2: ΔΔCₜ = ΔCₜ(Treated) – ΔCₜ(Control) = 2.5 – 4.5 = –2.0
  Step 3: Fold change = 2^(–ΔΔCₜ) = 2^(2.0) = 4
  Interpretation: The GOI is expressed 4‑fold higher in the treated sample compared with the control.

Common Sources of Error & Troubleshooting (AO3)

Linking PCR to Down‑stream Genetic Technology (AO2)

1. Restriction‑enzyme/ligase workflow – After PCR, the product is purified and digested with appropriate restriction enzymes (matching the sites added to the primers). The digested fragment is ligated into a plasmid vector that has been opened with the same enzymes, creating a recombinant DNA molecule ready for transformation.
2. Cloning – Ligated plasmid is introduced into a competent bacterial host (e.g., E. coli) by heat‑shock or electroporation. Colonies are screened for the insert (colony PCR, restriction analysis, or sequencing).
3. Site‑directed mutagenesis – Primers containing the desired base change amplify the whole plasmid; treatment with DpnI removes the parental (methylated) DNA, leaving only the mutated product for transformation.
4. Diagnostic testing – Pathogen‑specific primers amplify a unique gene fragment; presence/absence on a gel indicates infection.
5. Quantitative expression analysis – Reverse‑transcription PCR (RT‑PCR) converts mRNA to cDNA, which is then quantified by qPCR.
6. CRISPR‑Cas9 guide validation – PCR across the target site confirms successful editing (size shift, loss/gain of a restriction site, or sequencing of the amplicon).

Ethical, Social & Biosafety Considerations (AO3)

• GMO regulation – PCR‑derived clones can be used to create genetically modified organisms; students should be aware of national and international legislation (e.g., Cartagena Protocol).
• Pathogen detection – PCR is widely used for rapid diagnosis of infectious diseases; strict containment and decontamination procedures are required to prevent accidental release.
• Data privacy – When PCR is applied to human DNA (e.g., forensic or diagnostic testing), confidentiality and informed consent are essential.
• Environmental impact – Proper disposal of amplified DNA and reagents (especially ethidium bromide or other mutagens) must follow laboratory safety guidelines.

Why PCR Matters – Linking Back to the Syllabus

PCR demonstrates how DNA, the molecule of heredity, can be deliberately manipulated: a tiny fragment can be copied millions of times in a few hours, providing the raw material for cloning, sequencing, diagnostics and gene‑editing. Mastery of PCR therefore illustrates the key concepts of the “Genetic Technology” strand – the ability to isolate, amplify, modify and analyse genetic material – and highlights the societal responsibilities that accompany such powerful tools.

Key Points to Remember (Summary)

• Each PCR cycle doubles the target DNA; after 30 cycles >10⁹ copies are produced.
• Accurate primer design (18–25 nt, 40–60 % GC, T_m 55–65 °C, minimal secondary structure) is essential for specificity.
• Mg²⁺ concentration, annealing temperature, and cycle number must be optimised for each template.
• Taq polymerase is thermostable but lacks proofreading; choose a high‑fidelity polymerase when sequence accuracy is critical.
• Include appropriate positive, negative, and (where relevant) no‑polymerase controls to detect contamination and assess reaction success.
• Analyse products by agarose gel electrophoresis (size verification) or qPCR (quantitative data); interpret results against controls and expected band sizes.
• Common errors (no product, non‑specific bands, primer‑dimers) can usually be resolved by adjusting annealing temperature, Mg²⁺, primer concentration, or by using hot‑start enzymes.
• PCR is a gateway technique that links directly to restriction‑enzyme cloning, site‑directed mutagenesis, diagnostic testing, expression analysis and CRISPR validation, while raising important ethical and biosafety issues.