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Lesson 24 of 34 · The Polymerase Chain Reaction

PCR Principles and the Thermal Cycle

PCR Principles

The Goal: Amplifying a Defined Target

The polymerase chain reaction (PCR) makes many copies of one specific stretch of DNA. Starting from a sample that may contain only a few molecules of the sequence of interest buried among vast amounts of other DNA, PCR produces, in a few hours, enough copies of just that region to detect, measure, or analyze. It is amplification in vitro — performed in a tube with purified reagents rather than inside a living cell — and it is exponential: each round of copying roughly doubles the amount of the target 1.

The region that PCR copies is called the amplicon, and it is defined entirely by the reagents the operator chooses. PCR does not amplify “a gene” in the abstract; it amplifies precisely the segment bounded by the two short synthetic DNA pieces — the primers — that the operator supplies. Everything that follows is a consequence of that simple idea: tell the reaction where the target begins and ends, supply an enzyme that can copy it, and cycle the temperature so the copying repeats.

This lesson covers the chemistry and the thermal cycle that make amplification work. How to choose primers well is the subject of the primer design and optimization lesson; how to measure product as it accumulates is covered under real-time and RT-PCR; and the specialized formats — nested, multiplex, allele-specific, and digital PCR — appear later under PCR variations.

Reaction Components

A PCR reaction is assembled from a small, fixed set of ingredients. Each one connects to the polymerase requirements established earlier: a DNA polymerase needs a template, a primed 3’-hydroxyl, the four dNTPs, and magnesium, and it synthesizes in the 5’→3’ direction.

  • Template DNA — the sample containing the sequence to be copied. It need not be pure; the primers provide the specificity.
  • Two primers — short single-stranded oligonucleotides (typically ~18–25 nucleotides) that bind the template on opposite strands, flanking the target. They define both ends of the amplicon, and each provides the free 3’-OH that the polymerase extends.
  • dNTPs — a mixture of dATP, dCTP, dGTP, and dTTP, the nucleotide building blocks incorporated into each new strand.
  • A thermostable DNA polymerase — most commonly Taq polymerase, from Thermus aquaticus. Its heat stability, established in the polymerases lesson, is what lets a single addition of enzyme survive repeated near-boiling steps; an ordinary polymerase would be destroyed on the first cycle.
  • Buffer with magnesium (Mg2+) — maintains pH and ionic conditions, and supplies the divalent cation the polymerase requires for catalysis. Magnesium concentration is a key tunable variable in optimization 1.

These components are combined once, at the start. Because the polymerase is thermostable, nothing needs to be replenished between cycles, which is what makes the whole process automatable in a programmable heating block called a thermal cycler 2.

The Three Temperature Steps of a Cycle

Each PCR cycle is a sequence of three temperature steps. The temperatures below are typical and approximate; exact values are adjusted during optimization, especially the annealing temperature, which depends on the primers.

1. Denaturation (~94–95 °C). The reaction is heated to near boiling. At this temperature the hydrogen bonds holding the two strands of the DNA duplex together break, and the strands separate into single strands. This is the same melting of base pairing introduced under base pairing and hybridization, driven here on purpose by heat. Separating the strands is necessary because the polymerase copies a single-stranded template; it cannot read a strand still paired with its complement 3.

2. Annealing (~50–65 °C). The reaction is cooled so the primers can bind. At this lower temperature, complementary base pairing becomes favorable again, and the two primers anneal (hybridize) to their matching sequences on the now-separated template strands. The right annealing temperature is set just below the primers’ melting temperature (Tm): high enough that primers bind only their intended, fully complementary target, but low enough that they bind at all. This is where the Tm concept from the base pairing lesson becomes a practical design constraint 1.

3. Extension (~72 °C). The temperature is raised to the optimum for the thermostable polymerase. Starting from the free 3’-OH of each bound primer, the polymerase adds dNTPs complementary to the template, synthesizing a new strand in the 5’→3’ direction — exactly the reaction and requirements described in the polymerases lesson, now running at high temperature because the enzyme tolerates it. Each primer is thereby extended into a full copy of the strand it was bound to 3.

The three steps then repeat. One cycle takes only minutes, and a typical run is 25–40 cycles.

  One PCR cycle (temperatures are typical/approximate):

  ~94-95 C  DENATURATION   5'==============3'   duplex melts
                           3'==============5'   into single strands

  ~50-65 C  ANNEALING      5'==============3'
                              <===3' primer      primers bind
                3' primer===>                    flanking target
                           3'==============5'

  ~72 C     EXTENSION      5'==============3'
                              <====3'--------     polymerase
                ------3'====>                     extends 5'->3'
                           3'==============5'

  --> repeat 25-40 times; each cycle ~doubles the target

Why Two Primers in Opposite Orientation

A single primer would let the polymerase copy only outward in one direction, with no defined far end — the product would have no fixed length and would not double. PCR uses two primers, one for each strand, pointing toward each other. Because the polymerase always extends 5’→3’, a primer placed on the top strand and a primer placed on the bottom strand direct synthesis inward, so that each new strand runs across the region between the two primer sites.

The two primer binding sites therefore mark the two ends of the amplicon: the segment copied is precisely the stretch from one primer to the other, and copies made in later cycles are bounded at both ends by the primer sequences. Opposite orientation is what converts open-ended copying into a defined, doubling product 2.

  5'----[fwd primer]------------------------->----3'  top strand
  3'----<-------------------[rev primer]----------5'  bottom strand
        |<--------- amplicon (defined) --------->|

Exponential Amplification and the Three Phases

After the first few cycles, both newly made strands are themselves bounded by primer sites, so every copy can serve as a template in the next cycle. The number of target copies therefore grows as roughly 2^n, where n is the number of cycles: one molecule becomes 2, then 4, 8, 16, and so on. Twenty cycles of perfect doubling is about a millionfold increase; thirty cycles, about a billionfold 1.

Real reactions do not double forever. Plotted against cycle number, product accumulation passes through three phases:

  • Exponential phase — early cycles, when reagents are abundant and doubling is essentially complete each cycle. Product is proportional to starting target, which is why this phase is the basis for quantitative measurement (covered under real-time PCR).
  • Linear phase — efficiency falls and the increase per cycle is no longer a clean doubling.
  • Plateau phase — accumulation levels off and stops.

The plateau occurs because the reaction exhausts itself: primers and dNTPs are depleted, the polymerase loses activity over many high-temperature cycles, and the sheer concentration of product strands lets them re-anneal to each other faster than primers can bind, competing with the reaction. Once any of these limits is reached, more cycles add little product. Because plateau erases the proportional relationship between product and starting amount, quantitative methods read the signal during the exponential phase rather than at the end 2.

  product
    |                          ______ plateau
    |                       __/
    |                    __/  linear
    |                 _/
    |             __/  exponential
    |   ____----/
    +------------------------------------> cycle number

Summary

PCR amplifies a defined DNA target exponentially in vitro by repeating a three-step thermal cycle. Denaturation (~94–95 °C) melts the duplex into single strands; annealing (~50–65 °C) lets two primers hybridize to the sequences flanking the target, just below their Tm; extension (~72 °C) lets a thermostable polymerase such as Taq build new strands 5’→3’ from each primer’s 3’-OH, using the supplied dNTPs and Mg2+. Two primers in opposite orientation define both ends of the amplicon and turn open-ended copying into a doubling product. Copy number grows as roughly 2^n through an exponential phase, slows through a linear phase, and stops at a plateau when reagents and enzyme are exhausted. These principles underlie primer design, real-time and RT-PCR, and the PCR variations treated in the lessons that follow.

References

  1. Lela Buckingham. Molecular Diagnostics: Fundamentals, Methods, and Clinical Applications. 3rd ed. F.A. Davis Company. 2019. verified
  2. Michael R. Green, Joseph Sambrook. Molecular Cloning: A Laboratory Manual. 4th ed. Cold Spring Harbor Laboratory Press. 2012. verified
  3. Bruce Alberts, Rebecca Heald, Alexander Johnson, David Morgan, Martin Raff, Keith Roberts, Peter Walter. Molecular Biology of the Cell. 7th ed. W. W. Norton & Company. 2022. verified