Polymerase Chain Reaction Steps

Why This Matters

PCR is the workhorse of molecular genetics—it's how researchers, forensic scientists, and clinicians take a tiny amount of DNA and amplify it into millions of copies for analysis. You're being tested on your understanding of DNA replication mechanics, enzyme function, and the thermodynamics of nucleic acid interactions. Every step of PCR mirrors principles you've learned about how DNA behaves: hydrogen bonding between base pairs, the directionality of synthesis, and the temperature-dependent activity of enzymes.

Don't just memorize "heat it, cool it, copy it." Know why each temperature matters, what's happening at the molecular level during each phase, and how the components work together to achieve exponential amplification. When an exam question asks you to troubleshoot a failed PCR or predict what happens if you change a variable, you need to understand the underlying biochemistry—not just the protocol.

---

The Three Core Cycling Steps

These are the heart of PCR—three temperature-dependent phases that repeat to amplify your target DNA. Each step exploits a specific property of DNA structure or enzyme kinetics.

Denaturation

• Temperature: 94-98°C—high heat breaks the hydrogen bonds between complementary base pairs, separating double-stranded DNA into two single strands
• Molecular mechanism: The energy input overcomes the hydrogen bonding and base stacking interactions that stabilize the double helix
• Why it matters: Single-stranded templates are required for primers to access their binding sites; incomplete denaturation leads to failed amplification

Annealing

• Temperature: 50-65°C—cooler conditions allow primers to hydrogen-bond to their complementary sequences on the template strands
• Temperature specificity: The exact annealing temperature depends on the melting temperature ($T_m$) of your primers; too high and primers won't bind, too low and they'll bind nonspecifically
• Directionality matters: Primers bind to the 3' end of each template strand, positioning DNA polymerase to synthesize in the 5'→3' direction

Extension

• Temperature: 72°C—the optimal activity temperature for Taq polymerase, which synthesizes new DNA by adding dNTPs to the 3' end of primers
• Synthesis direction: DNA polymerase can only add nucleotides to a free 3'-OH group, extending the primer toward the 5' end of the template
• Product formation: Each extension creates a new double-stranded DNA molecule; by the end of this step, you've doubled your target sequences

---

Amplification Dynamics

Understanding how PCR achieves exponential amplification is essential for predicting yields and troubleshooting problems.

Repetition of Cycles

• Cycle number: 20-40 cycles—each complete cycle (denaturation → annealing → extension) theoretically doubles the amount of target DNA
• Exponential amplification: After $n$ cycles, you have approximately $2^n$ copies of your target; 30 cycles yields over 1 billion copies from a single template
• Plateau phase: Amplification eventually slows as dNTPs deplete, polymerase degrades, and products begin to reanneal to each other instead of primers

Temperature Cycling

• Precision is critical—thermal cyclers must hit exact temperatures and hold them for specific durations to ensure complete denaturation, efficient annealing, and full extension
• Ramp rates: The speed of temperature transitions affects efficiency; too slow wastes time, too fast may cause incomplete phase transitions
• Cycle timing: Extension time depends on amplicon length—longer products require more time for polymerase to complete synthesis (roughly 1 minute per 1 kb for Taq)

---

Reaction Components

The success of PCR depends on carefully balanced reagents. Each component plays a specific biochemical role.

Thermostable DNA Polymerase

• Taq polymerase (from Thermus aquaticus) remains active at 95°C because its protein structure evolved for hot spring environments
• Fidelity trade-off: Taq lacks 3'→5' exonuclease proofreading activity, introducing approximately 1 error per $10^4$ bases; high-fidelity polymerases are used when accuracy is critical
• Processivity: The enzyme's ability to remain attached to the template affects how efficiently it synthesizes long products

dNTP Addition

• Building blocks: Equal concentrations of dATP, dTTP, dCTP, and dGTP (typically 200 µM each) provide the raw materials for new strand synthesis
• Concentration balance: Unequal dNTP ratios increase misincorporation errors because polymerase may insert incorrect nucleotides when the correct one is limiting
• Magnesium dependency: dNTPs chelate $Mg^{2+}$, so dNTP concentration directly affects the free magnesium available for polymerase activity

Buffer and Salt Conditions

• Buffer function: Tris-HCl (pH ~8.4 at 25°C) maintains optimal pH for enzyme activity; note that Tris pH drops at higher temperatures
• Salt effects: $KCl$ (50 mM) and $MgCl_2$ (1.5-2.5 mM) stabilize primer-template interactions and are essential cofactors for polymerase
• Optimization: Adjusting $Mg^{2+}$ concentration is often the first troubleshooting step—too little reduces yield, too much increases nonspecific amplification

---

Preparation and Design Elements

Before you even start cycling, proper preparation determines whether your PCR will succeed or fail.

Primer Design

• Length: 18-25 nucleotides—short enough for specific binding, long enough to ensure unique target recognition in complex genomes
• GC content: 40-60%—balanced composition ensures appropriate $T_m$ and stable binding; primers ending in G or C ("GC clamp") improve 3' end stability
• Avoid secondary structures: Primers that form hairpins or primer-dimers will self-anneal instead of binding template, drastically reducing amplification efficiency

Template DNA Preparation

• Purity matters: Contaminants like phenol, ethanol, or proteins inhibit Taq polymerase; A260/A280 ratios of 1.8-2.0 indicate clean DNA
• Quantity range: Typical reactions use 1-100 ng of genomic DNA or 0.1-1 ng of plasmid; too much template increases nonspecific products
• Integrity: Degraded or sheared template DNA may lack intact primer binding sites, leading to failed or shortened amplification

---

Quick Reference Table

---

Self-Check Questions

1. If you increased the annealing temperature above your primer $T_m$, what would happen to your PCR products and why?

2. Compare denaturation and annealing: both involve hydrogen bonding between nucleotides, but how does temperature manipulation achieve opposite outcomes in each step?

3. After 25 cycles of PCR starting with a single template molecule, approximately how many copies of the target sequence would you have? What assumption does this calculation make?

4. A researcher's PCR produces multiple bands of different sizes. Which two reaction components would you adjust first, and what changes would you make?

5. Contrast Taq polymerase with the DNA polymerase III used in bacterial replication—what key proofreading difference exists, and when would this matter for experimental outcomes?