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PCR and RT-PCR

PCR copies a chosen stretch of DNA over and over, doubling it in each cycle under ideal conditions. RT-PCR adds a first step that turns RNA into DNA so it can be amplified the same way.

The cycle

Each cycle separates the DNA strands with heat, lets short primers attach at a lower temperature, and then lets the polymerase extend them. Repeating this a few dozen times turns a trace of template into something easily detectable.


Primers decide specificity

Primers that match more than one place in the template, or match each other, produce extra bands and primer dimers. Most specificity problems are primer-design problems rather than reagent problems.


Controls

A no-template control shows whether something is amplifying that should not be. A positive control shows the reaction can work at all. Without both, a result of any kind is hard to interpret.


Quantitative PCR

Real-time or quantitative PCR follows the reaction as it happens and estimates how much starting material there was from how early the signal appears. It is sensitive to pipetting accuracy and to inhibitors carried over from extraction.

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