BACKYARD BIOLOGY
Running PCR
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).
