---------------------

by Shawn Carlson
July, 2000

---------------------

Mark my words: one day Eva Harris will win the Nobel Peace Prize. This visionary professor at the University of California at Berkeley will certainly deserve such recognition for her work, which could save countless lives. Harris develops inexpensive ways to conduct sophisticated biomedical tests and then brings that technology to people in the developing world. By providing the right equipment and training to local public health workers, she is building epidemiological firewalls around disease "hot spots." These preparations are now helping to contain outbreaks before they grow into epidemics.

In 1998 Harris founded the Sustainable Sciences Institute in San Francisco to carry out this mission, and already her group has achieved some stunning successes. As part of that effort, Harris recently published A Low-Cost Approach to PCR (Oxford University Press; ISBN: 0-19-511926-6), which is the definitive manual on cost-conscious biotech. Though intended for health professionals, this book is a boon for amateurs working on a budget. It explains how anyone with a bit of inexpensive equipment can carry out the polymerase chain reaction (PCR), a technique for generating large quantities of DNA.

Gene amplification begins with double-stranded DNA (a). Heat parts the strands (b), and short segments of DNA (primers) attach to specific locations (c). The polymerase enzyme attaches DNA building blocks (dNTPs, shown in yellow, green, pink and purple) sequentially to each strand, forming two new strings of DNA that complement the originals. Repetition of these steps doubles the amount of DNA present after each iteration. Click image to enlarge.

The PCR method unzips a DNA double helix into two complementary strings, which are immersed in a soup of DNA building blocks. The proper experimental conditions induce these constituents to assemble two new copies from what was originally one DNA molecule. The steps involved take just a few minutes. And repeating the procedure doubles the number of copies each time. So 30 cycles of PCR produce a billion-fold increase of the targeted section of DNA, "amplifying" what might begin as a single molecule into enough material for easy examination.

Amateur scientists can do PCR at home, but the exercise is quite challenging. For one, the very sensitivity of PCR means that this technique is extremely vulnerable to contamination: a single wayward cell could render your experiment meaningless. The serious experimenter should purchase Harris's book and a good textbook on biochemistry. To get you started, this column describes a demonstration of PCR that avoids most of the pitfalls. And the Society for Amateur Scientists can supply the materials that are difficult to obtain.

First, you will need some of your own DNA and several sterile Pyrex test tubes with rubber stoppers--or better yet, some plastic microcentrifuge tubes with built-in caps. You can reduce the risk of contamination by washing your glassware and working surface with bleach and by wearing latex gloves at all times. To collect the DNA sample, gently scrape the inside of your cheek with a sterile cotton swab, then slosh the tip around inside a clean tube filled with a few milliliters of distilled water. Gently boil the water for two minutes to rip open the cell walls and release your genetic blueprint. The solution will now contain a few DNA fragments, as well as other large molecules and sundry leftovers from the ruptured cells.

Let this biological broth cool and then, if you can, use a blender-centrifuge [see The Amateur Scientist, January 1998] to separate and remove the larger cellular debris. Some of the dissolved molecules can interfere with PCR, so practitioners usually dilute the solution by factors of 10 and 100 to reduce the concentration of any troublesome ingredients. Once you have made these preparations, keep your samples packed in ice until you are ready to use them.

The high price of materials leads even professionals to use fantastically tiny amounts of the various reagents, often one microliter or less. Dishing out such small quantities typically requires a calibrated pipetting tool (such as part no. S346503 from Fisher Scientific, $219; you'll also need the disposable pipette tips, part no. S346501, which cost about $30 for a set). But you can instead employ translucent plastic coffee stirrers. Just dip the straw into the solution to the appropriate depth and cover the end with your thumb as you transfer the contents. The set of white stir sticks I purchased from my grocery store cost less than two cents apiece and yet deliver about 70 microliters for each centimeter of length. I found that I could transfer 70 microliters of liquid very consistently (to within about 4 percent), and I could dole out as little as five microliters with only about 40 percent error.

RECIPE FOR PCR SOUP requires many ingredients mixed together in the specified relative proportions, but only the concentration of magnesium chloride is truly critical.

The recipe for PCR soup given above consists of a buffer, two primers, a polymerase enzyme, DNA building blocks called deoxynucleotide triphosphates, or dNTPs (see below) 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.

Polymerase Chain Reaction by Elizabeth A. Dragon Roche Molecular Systems Polymerase chain reaction (PCR) is a technique that mimics nature's way of replicating DNA. First described in 1985, PCR has been adopted as an essential research tool because it can take a minute sample of genetic material and duplicate enough of it for study. PCR has been used to identify the remains of Desert Storm casualties, to analyze prehistoric DNA, to diagnose diseases and to help make identifications in police investigations. DNA is most often found as a double-stranded molecule, twisted as a helix, in which each strand complements the other. PCR starts with the DNA sample, which is put in a reaction tube along with primers (short, synthetic pieces of single-stranded DNA that exactly match and flank the stretch of DNA to be amplified), deoxynucleotide triphosphates (dNTPs, the building blocks of DNA), buffers and a heat-resistant enzyme (polymerase). Heating the mixture separates the "template" strands of DNA. Then, at varying temperatures, the rest of the components in the mixture spontaneously organize themselves, building a new complementary strand for each original. At the end of each cycle the DNA count has doubled. If you start with one DNA molecule, at the end of 30 cycles (only a few hours later) there will be about a billion copies. Thus, if you are looking for a single gene among thousands, the game changes from "searching for a needle in a haystack" to "making a haystack of needles." DUPLICATING DNA begins with a double-stranded stretch of DNA to be amplified, or copied (a). In a solution heated to 95 degrees Celsius (203 degrees Fahrenheit), hydrogen bonds between the strands break, leaving two single strands (b). When the mixture is cooled to between 50 and 65 degrees C, specially manufactured DNA primers bind complementarily to each strand at points flanking the region to be copied (c). At 72 degrees C, polymerase enzymes extend the bound primers in one direction, using the original DNA as a template (d). The products are two new double strands of DNA, both identical to the original (e). This cyclic reaction takes only minutes or less and can be repeated indefinitely. Click image to enlarge. POLYMERASE ENZYME extends a bound primer. From the surrounding medium, it extracts a free-floating deoxynucleotide triphosphate (dNTP) that will complement the next unpaired position in the template strand of DNA. The enzyme then joins the dNTP to the end of the primer and moves on to the next position. EXPONENTIAL GROWTH of the DNA target occurs because the products of each cycle become the templates for the next cycle. Click image to enlarge.

The three heating steps can be simply carried out by arranging three hot water baths and transferring the tubes among them. I just put pots of water on my stove and monitored their temperatures using candy thermometers. It took three hours to shepherd my samples through the baths 30 times. I used a thermocouple inside one of my test tubes to check how quickly the solution reached the proper temperature (one to two minutes); tiny microcentrifuge tubes will equilibrate much faster.

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.

The Society for Amateur Scientists
5600 Post Road, #114-341
East Greenwich, RI 02818
Phone: 1-401-823-7800

Internet: http://www.sas.org/

American Science & Surplus offers a unique mix of industrial, military and educational items, with an emphasis on science and education. We supply a wide range of unusual and hard to find items (some say bizarre stuff) to the hobbyist, tinkerer, artist, experimenter, home educator, do-it-yourselfer, and bargain hunter.

American Science & Surplus
P.O. Box 1030
Skokie, IL 60076
847-982-0870 Voice
800-934-0722 Fax

info@sciplus.com

http://www.sciplus.com