PCR Principles and the Thermal Cycle

The goal

PCR copies one defined stretch of DNA - the amplicon - millions to billions of times, so a few starting molecules become enough to detect, size, or sequence ¹. It is DNA replication stripped to the polymerase’s requirements (see the Foundations lesson on replication).

What goes in the tube

Master mixes premix everything except primers and template. Every reaction set includes positive, negative, and no-template controls (see Assay Design).

One cycle, three temperatures

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A run is typically 25-40 cycles, often finished with a final extension.

Two primers, one product

The primers bind opposite strands with their 3’ ends pointing toward each other ¹. In early cycles, products copied from genomic template run past the far primer site; but from the third cycle on, strands appear that begin at one primer and end at the other. These defined-length products are themselves templates and soon dominate: the amplicon is exactly primer to primer.

Doubling and its limits

Each perfect cycle doubles the target:

• 2^10 ≈ 1,000; 2^20 ≈ 1 million; 2^30 ≈ 1 billion.
• 100 starting copies after 30 cycles: about 10^11.

Real reactions run at somewhat under 100% efficiency, and they go through three phases ¹:

1. Exponential: reagents in excess, near-perfect doubling; product is proportional to starting amount.
2. Linear: primers, dNTPs, or enzyme start to run short; gains slow.
3. Plateau: little more product, and products re-anneal to each other instead of to primers.

Because reactions with 10 or 10,000 starting copies can both reach plateau, the endpoint yield says little about starting amount - which is why quantitative PCR measures during the exponential phase (see Real-Time PCR). Plateau is also when contamination becomes dangerous: a tube now holds enough amplicon to contaminate a whole lab with a drop.

What to take from this

PCR needs template, two primers, dNTPs, a thermostable polymerase, and Mg²⁺ buffer. Each cycle denatures (about 95 °C), anneals (about 55-65 °C), and extends (72 °C), and two inward-pointing primers make a product that runs exactly primer to primer. Doubling means 2^30 ≈ a billion copies, but reactions plateau, so only exponential-phase measurement quantifies.