The recipe for PCR soup given above consists of a buffer, two primers, a polymerase enzyme, DNA building blocks (called \deoxynucleotide triphosphates, or dNTPs) and magnesium chloride. The buffer keeps the reaction at a constant pH. The primers are short fragments of unzipped DNA that bond to the specific sites on human DNA and define where the copying begins and ends. The polymerase enzyme assembles the DNA building blocks, and the magnesium in the solution helps keep the reaction going.
Make up several tubes with these ingredients. Be certain that one tube contains only the reagents; that is, do not add any of your DNA to it. You will run this one through the amplification steps to serve as a negative control: no DNA should show up in this vial in the end.
Begin the PCR cycle by splitting the DNA with heat. At about 94 degrees Celsius (201 degrees Fahrenheit), the double helix unravels in roughly a minute. You should keep your test tubes stoppered (or your microcentrifuge tubes capped) to prevent evaporation. Next, lower the temperature to about 60 degrees C (140 degrees F) for about 90 seconds. This step induces the primers to bond to the separated DNA strings. Then raise the temperature to 72 degrees C (162 degrees F) for another 90 seconds, allowing the heat-hardy polymerase (an enzyme that comes from a bacterium native to hot springs) to build the new copies.
You should end up with loads of DNA molecules, which you can sort by size using gel electrophoresis [see The Amateur Scientist,December 1998]. During my tests, I ran three dilutions and one negative control. A more sophisticated researcher would also include a calibration solution that contains DNA fragments of known lengths. Comparing results with the calibration solution makes it easy to gauge the size of the amplified DNA.
After running my electrophoresis gel at 54 volts (generated with six nine-volt batteries) for an hour, I stained it with a dilute solution of ethidium bromide--a nasty mutagenic chemical, which can be absorbed directly through the skin, so take great care not to get any on yourself. Ethidium bromide bonds directly to DNA and fluoresces when illuminated with ultraviolet (UV) light. I darkened my bathroom and used an ordinary (long-wave) black light to observe the faint lines of amplified DNA. Experimenters using a short-wave UV light will see much brighter lines. These so-called transilluminators cost $195 from Fisher Scientific (part no. S45157). But remember that when working with short-wave UV, you must wear UV-protective goggles (such as part no. S47733 from Fisher Scientific, $7) whenever the light is on to avoid damaging your eyes. If you have any doubts about how vigilant you can be, just stick with an ordinary black light.
The ability to do PCR at home opens vast new territories for amateur exploration. If you get good at applying this technique, you might even be able to help the Sustainable Sciences Institute stem the spread of disease. In any case, I urge you to find out more about this wonderful group, which I am sure will eventually receive the widespread praise and support it merits. It took the Nobel committee almost three decades to award the prize to the French humanitarian organization Doctors Without Borders. I just hope that Eva Harris and her colleagues will not have to wait so long
This article was originally published with the title PCR at Home.