DIYbio in 2000
Reshma Shetty
Amateur Scientist column on doing PCR at home, from May 2000
http://www.sciam.com/article.cfm?id=pcr-at-home
[Text included below.] There's also an article on gel electrophoresis
at home from 1998 referenced in the article, but I don't have access
to it. Same with this article re the end of DIY :)
http://www.sciam.com/article.cfm?id=rip-for-diy
Also, on another recent topic, I like the name DIY biological engineering.
-Reshma
PCR AT HOME
SHAWN CARLSON
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.
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
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.
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
Bryan Bishop
> Just came across this article from Scientific American's now defunct
> Amateur Scientist column on doing PCR at home, from May 2000
>
> http://www.sciam.com/article.cfm?id=pcr-at-home
>
> [Text included below.] There's also an article on gel electrophoresis
> at home from 1998 referenced in the article, but I don't have access
> to it. Same with this article re the end of DIY :)
> http://www.sciam.com/article.cfm?id=rip-for-diy
From my link collection on PCR :-)
http://www.sciam.com/article.cfm?articleID=00035C6C-229B-1C74-9B81809EC588EF21
http://pathmicro.med.sc.edu/pcr/realtime-home.htm
http://humgen.wustl.edu/hdk_lab_manual/pcr/pcr1.html
http://www.protocol-online.org/prot/Molecular_Biology/PCR/
http://www.how-2-diy.com/tag/pcr
http://www.sumanasinc.com/webcontent/animations/content/pcr.html
http://pathmicro.med.sc.edu/pcr/realtime-home.htm
From my other stuff for home-done PCR, see-
http://heybryan.org/~bbishop/docs/ellingtonia/biotech/
Norman Wang
May2002, Vol. 286 Issue 5, p26, 1p), text below:
http://www.sciam.com/article.cfm?id=rip-for-diy
---
Section: news SCAN
AMATEUR SCIENCE
SCIENCE TINKERERS CONTINUE TO TAKE IT ON THE CHIN
Several years ago I walked into Fry's Electronics in Palo Alto,
Calif., and asked for an inductor. It is hardly an unusual electronic
component; every radio project needs one. Yet the store clerks looked
at me blankly. Fry's once had a reputation as the first stop for young
engineers stocking a garage workshop. But in its components aisle, I
found just a few bags of parts.
"The art of home-brewing one's own electronic equipment is pretty much
a lost one," says Chuck Penson, a radio ham in Tucson, Ariz. The
D.I.Y. movement that spawned the computer revolution--and inspired
untold numbers of tinkerers to pursue careers in science--has stopped
moving. Heathkit ceased making its electronic kits 10 years ago.
Popular Electronics and Byte magazines have hung up their soldering
irons. Meccano, the maker of Erector sets, went bankrupt in 2000. Last
year Scientific American dropped the Amateur Scientist column, citing
a long decline in readership, and Edmund Scientific sold off its
consumer catalogue and shut its famous retail store in Barrington,
N.J.
"It was a Mecca for the science enthusiast," recalls Nicole Edmund,
vice president of marketing and sales at Edmund Industrial Optics and
granddaughter of the company's founder. But the store's sales had been
drooping for most of the past decade, she says, and the company wanted
to focus on its more profitable optics business.
What we seem to have witnessed is the fragmentation of amateur
science. Heathkit, for example, appealed to a broad range of people.
Some built kits for kits' sake. Others just wanted to save money:
Heathkits were usually cheaper and better than store-bought radios or
TVs. As manufacturing costs went down and quality went up, though, off-
the-shelf products gained the advantage. The same went for telescopes
and most other gizmos. "When I got started, I could not have purchased
what I could have built," says Dennis DiCicco, an editor at Sky &
Telescope magazine. "Today if you want a telescope, you can afford
one. You're not going to save much money if you build one."
As the market split between craftsmen and appliance owners, magazines
had to adapt or die. In the late 1970s computer hobbyists of all
ability levels devoured Byte. As PCs went mainstream, the magazine
played down home-brew projects. Advanced amateurs, meanwhile, outgrew
the projects and gravitated to niche publications. Circuit Cellar,
started by ex-Byte columnist Steve Ciarcia, succeeded with a new
publishing model: as its readers became more sophisticated, so did the
articles. "I saw that you had to move upscale with them, or they'd
move away from you," Ciarcia says.
Indeed, dedicated amateurs are now quasi-professionals. The Society
for Amateur Scientists conference taking place next month in
Philadelphia will have sessions on how to publish your research and
how to claim a tax deduction for your basement lab. Discoveries by
amateur astronomers have made headlines. At the other end of the
market, people with an occasional science craving can satisfy it at,
say, the Nature Company. And for those who fall in the middle, a few
kit suppliers (especially in robotics and music production) and
magazines (such as Nuts & Volts and Poptronics, formerly Radio
Electronics) carry on.
Evidently, the something-for-everyone model epitomized by Heathkit and
the Amateur Scientist column can't compete anymore. Specialized
sources and Internet newsgroups cater to each skill level. But much of
the mentoring and serendipity that the diverse community of amateurs
offered has been lost. It is hard not to regret its passing.
PHOTO (COLOR): HAM RADIO remains a vibrant hobby, as is evident at
flea markets such as this "Hamfest" in Clinton, N.J. But after a
growth spurt in the 1990s, the number of amateur radio enthusiasts is
starting to drop.