You perform polymerase chain reaction (PCR) by mixing your sample with polymerase reagent and primers in a small test tube, then running it repeatedly through a sequence of temperature changes in your thermal cycler. Each temperature change facilitates a different step in the replication process, and the entire sequence doubles the volume of DNA in the tube that’s identical to the original sample. Repeat the process 30 or 40 times, and you’ll end up with a billion or trillion-fold increase in your DNA, as long as your primer molecules hold out.
Gel electrophoresis, as outlined in the first part of this article, determines the DNA’s “fingerprint,” and the large amounts of DNA from PCR make these fingerprints easy to match. Run identical gel electrophoresis on generously replicated samples, and if they produce a similar set of lines on the gel, then the samples match — although it’s still possible (if unlikely) that the matching lines come from different DNA strands with the same lengths and weights.
1. Open the DNA.
(Thermal cycle: about 95ºC for 1 minute) DNA exists as a tight helical coil, and applying a high temperature unravels it.
DNA
Heat to open
2. Primers bind.
(Thermal cycle: about 60ºC for 1 minute) With the DNA open, shorter pieces of DNA called primers — designed to interact with a region you wish to copy — can now get in and bind to each half. The second temperature encourages this binding reaction.
Primers
Primers binding
3. Polymerase extends replication. (Thermal cycle: about 70ºC for 1-3 minutes) With the primers bound and providing a foothold, the heat-stable DNA polymerase runs along the half-DNA original and extends replication of the other half. This is the actual “replication” part, and the new temperature optimizes enzyme function. At the end of this step, you have two copies of the DNA strand of interest, and both return to their original helical form.
Polymerase
Polymerase attaches ....
... and extends
The Advanced Molecular Biology Lab at the University of British Columbia likes to play with and teach about DNA, RNA, and proteins. They publish the Science Creative Quarterly ( scq.ubc.ca).
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