Biology · Introductory biology · Concept

PCR: the polymerase chain reaction

The polymerase chain reaction (PCR) copies one chosen stretch of DNA millions of times. Two short primers match the two ends of the target, and each cycle of denaturation, annealing and extension can double the number of copies, so n cycles multiply the starting amount by up to 2ⁿ. The product, the amplicon, runs from one primer to the other.

One cycle, three steps

Heating to about 95 °C separates the two DNA strands (denaturation). Cooling to roughly 50–65 °C, depending on the primers, lets each primer bind its matching sequence (annealing). At about 72 °C a heat-stable DNA polymerase extends each primer by adding nucleotides to its 3′ end (extension). A cycle takes a few minutes.

Primers choose the product

The forward primer has the same sequence as the top strand at the left end of the target, and it binds the bottom strand. The reverse primer binds the top strand at the right end, so its sequence, written 5′→3′, is the reverse complement of the top strand there. The primers extend toward each other, and the product runs from the 5′ end of one to the 5′ end of the other, including both primer sequences.

Doubling every cycle

If every copy is copied in each cycle, the number of copies doubles: n cycles multiply the starting number N₀ by 2ⁿ. With efficiency E, the fraction of copies duplicated each cycle, the factor per cycle is 1 + E. Thirty perfect cycles turn one molecule into 2³⁰, about 1.07 billion.

N = N_{0} {(1 + E)}^{n}

Why real reactions level off

Growth is exponential only in the early cycles. Primers and nucleotides get used up, the polymerase loses activity, and product strands pair with each other instead of with primers, so the reaction reaches a plateau. The doubling model describes the exponential phase, not the whole run.

The first few cycles

Copies made from the original long template in the first cycles run past the target at one end. Products of exactly the target length first appear in cycle 3, and after that they quickly outnumber every other product.

What PCR needs and what it does

Primers require known sequence at both ends of the target. With them, PCR detects the DNA of a pathogen, copies a gene for cloning, and amplifies the tiny amounts of DNA in forensic samples. It copies contaminating DNA just as well, which is why labs run negative controls.

Common mistakes

Writing the reverse primer as the top-strand sequence at the right end instead of its reverse complement.
Leaving the primer sequences out of the product length: the amplicon includes both.
Expecting doubling to continue indefinitely: late cycles plateau as reagents run out.
Confusing cycles with copies: 30 perfect cycles give 2³⁰ copies, not 60.

Key terms

Polymerase chain reaction: A lab method that copies one chosen stretch of DNA millions of times through repeated heating and cooling cycles, guided by a pair of primers. This app models the sequence matching and ideal copying, not a lab protocol.
Primer: A short piece of DNA that binds next to the region to copy and gives the polymerase a starting point. A PCR primer pair binds opposite strands, facing each other.
Amplicon: The piece of DNA that PCR copies: the stretch between and including the two primer sites.
DNA denaturation: Separation of double-stranded DNA into single strands as the hydrogen bonds between base pairs break, as when PCR heats DNA to about 95 °C.
Primer annealing: The step where primers bind to their matching sequences on the template as the reaction cools. A matching sequence alone doesn’t guarantee binding at every temperature.
Amplification efficiency: How much the DNA increases per PCR cycle: an efficiency of 1 (100%) means exact doubling. Real reactions usually run a little below that and slow down in later cycles.
Reverse complement: The sequence of the opposite DNA strand, read 5′ to 3′: swap each base for its partner (A↔T, C↔G) and reverse the order. For 5′-ATGC-3′ it is 5′-GCAT-3′.

Work through an example

The top strand of a template reads 5′-CCTAGATCGT ACCATTGGCA TCGACTAGGC TTCACGGGAT-3′, 40 bases shown in blocks of 10. The forward primer is 5′-GATCGTACCA-3′ and the reverse primer is 5′-CGTGAAGCCT-3′. Find the product and its length, and the number of copies 30 ideal cycles make from one template.

Find the PCR product from two primers →

Calculate DNA copies after PCR cycles →

Sources and scope

Authored study material. Tool results depend on the stated inputs and model assumptions.

Make it concrete

Try in the workspace

Open the example inputs, change a value and keep a useful result on your board.

Open the PCR tool Open worked example on a board Molecular lab reference

Your existing work stays on this device. Examples open as editable copies.