Adv Physiol Educ 28: 44 –50, 2004;

Staying Current 10.1152/advan.00002.2004.

The polymerase chain reaction

Tabitha M. Powledge
Tabitha Powledge is a freelance science writer.

Powledge, Tabitha M. The polymerase chain reaction. Adv the enzymes known as polymerases. These enzymes are
Physiol Educ 28: 44 –50, 2004; 10.1152/advan.00002.2004.—This present in all living things, and their job is to copy genetic
essay on the polymerase chain reaction is one of a series developed as material (and also proofread and correct the copies). Some-
part of FASEB’S efforts to educate the general public, and the
times referred to as “molecular photocopying,” PCR can char-
legislators whom it elects, about the benefits of fundamental biomed-
ical research—particularly how investment in such research leads to acterize, analyze, and synthesize any specific piece of DNA or
scientific progress, improved health, and economic well-being.1 RNA. It works even on extremely complicated mixtures, seek-
ing out, identifying, and duplicating a particular bit of genetic
material from blood, hair, or tissue specimens, from microbes,
IT IS HARD TO EXAGGERATE THE IMPACT of the polymerase chain animals, or plants, some of them many thousands— or possibly
reaction. PCR, the quick, easy method for generating unlimited even millions— of years old.
copies of any fragment of DNA, is one of those scientific So simple is the PCR process, at least to molecular biolo-
developments that actually deserves time-worn superlatives gists, that its inventor, Kary Mullis, says their universal reac-
like “revolutionary” and “breakthrough.” tion has always been, “Why didn’t I think of that?” Among the
First described only 10 years ago, in its short life PCR has host of scientific prizes heaped on Mullis for the very bright
transformed the life sciences utterly. From the daily practicali- idea he says came to him during a 1983 moonlight drive in the
ties of medical diagnosis to the theoretical framework of sys- California mountains are two of the best known, the Japan
tematics, from courts of law to field studies of animal behavior, Prize and the Nobel, both awarded to him in 1993.
PCR takes analysis of tiny amounts of genetic material— even PCR requires a template molecule—the DNA or RNA you
damaged genetic material—to a new level of precision and want to copy—and two primer molecules to get the copying
reliability. process started. The primers are short chains of the four dif-
“PCR is the most important new scientific technology to ferent chemical components that make up any strand of genetic
come along in the last hundred years,” says Mark R. Hughes, material. These four components are like bricks or building
Deputy Director of the National Center for Human Genome blocks that are used to construct genetic molecules; in the lab
Research at the National Institutes of Health (perhaps better they are called nucleotides or bases.
known as the Human Genome Project). And Science has DNA itself is a chain of nucleotides. Under most conditions,
pointed out that, because it is far simpler and less expensive DNA is double stranded, consisting of two such nucleotide
than previous techniques for duplicating DNA, PCR has de- chains that wind around each other in the famous shape known
mocratized genetic research, putting it within reach of all as the double helix. Primers are single-stranded. They consist
biologists, even those with no training in molecular biology. of a string of nucleotides in a specific order that will, under the
right conditions, bind to a specific complementary sequence of
WHAT IS PCR? nucleotides in another piece of single-stranded RNA or DNA.
For PCR, primers must be duplicates of nucleotide se-
The central scientific fact that makes PCR so useful is this:
the genetic material of each living organism—plant or animal, quences on either side of the piece of DNA of interest, which
bacterium or virus—possesses sequences of its nucleotide means that the exact order of the primers’ nucleotides must
building blocks (usually DNA, sometimes RNA) that are already be known. These flanking sequences can be con-
uniquely and specifically present only in its own species. structed in the lab, or purchased from commercial suppliers.
Indeed, complex organisms such as human beings possess There are three basic steps in PCR. First, the target genetic
DNA sequences that are uniquely and specifically present only material must be denatured—that is, the strands of its helix
in particular individuals. These unique variations make it pos- must be unwound and separated— by heating to 90 –96°C (Fig.
sible to trace genetic material back to its origin, identifying 1). The second step is hybridization or annealing, in which the
with precision at least what species of organism it came from, primers bind to their complementary bases on the now single-
and often which particular member of that species. stranded DNA. The third is DNA synthesis by a polymerase.
Such an investigation requires, however, that enough of the Starting from the primer, the polymerase can read a template
DNA under study is available for analysis—which is where strand and match it with complementary nucleotides very
PCR comes in. PCR exploits the remarkable natural function of quickly. The result is two new helixes in place of the first, each
composed of one of the original strands plus its newly assem-
bled complementary strand.
Address for reprint requests and other correspondence: D. U. Silverthorn, All PCR really requires in the way of equipment is a reaction
Neurobiology Section, Univ. of Texas, Austin, TX 78712 (E-mail: tube, reagents, and a source of heat. But different temperatures
silverthorn@mail.utexas.edu). are optimal for each of the three steps, so machines now
1
This article was first published in 1995 as part of a series, Breakthroughs
in Bioscience, by the Federation of American Societies for Experimental
control these temperature variations automatically.
Biology on FASEB’s Public Policy Home Page (http://www.faseb.org/opar/ To get more of the DNA you want, just repeat the process,
opar.html). It is republished here with the kind permission of FASEB. beginning by denaturing the DNA you’ve already made. The
44
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POLYMERASE CHAIN REACTION 45

HOW IS PCR USED?

Human Health and the Human Genome Project

PCR has very quickly become an essential tool for improv-
ing human health and human life (Fig. 2). Medical research and
clinical medicine are profiting from PCR mainly in two areas:
detection of infectious disease organisms, and detection of
variations and mutations in genes, especially human genes.
Because PCR can amplify unimaginably tiny amounts of DNA,
even that from just one cell, physicians and researchers can
examine a single sperm, or track down the elusive source of a
puzzling infection. These PCR-based analyses are proving to
be just as reliable as previous methods—sometimes more so—
and often much faster and cheaper.
The method is especially useful for searching out disease
organisms that are difficult or impossible to culture, such as
many kinds of bacteria, fungi, and viruses, because it can
generate analyzable quantities of the organism’s genetic mate-
rial for identification. It can, for example, detect the AIDS
virus sooner, during the first few weeks after infection, than the
standard ELISA test. PCR looks directly for the virus’s unique
Fig. 1. How primers work (from Recombinant DNA, 2nd edition, by Watson
DNA instead of the method employed by the standard test,
JD, Gilman M, Witkowaski J, and Zoller M. Copyright © 1992 Watson JD, which looks for indirect evidence that the virus is present by
Gilman M, Witkowaski J, and Zoller M. Used with permission of WH Freeman searching for antibodies the body has made against it.
and Co). PCR can also be more accurate than standard tests. It is
making a difference, for example, in a painful, serious, and
often stubborn misfortune of childhood, the middle ear infec-
amount will double every time. With the cycle of rapid heating
and cooling controlled automatically, nature-aided by scientist-
supplied primers, polymerase, nucleotides, and chemical re-
agents does the rest. Each cycle takes only 1–3 minutes, so
repeating the process for just 45 minutes can generate millions
of copies of a specific DNA strand. Once the primers have been
characterized and obtained, PCR can do in a week’s work what
used to take a year.
Of course, some technical problems can arise with PCR. The
most important is contamination of the sample with extraneous
genetic material that could generate numerous copies of irrel-
evant DNA. The result will often simply be useless, but some-
times can lead to erroneous conclusions. Laboratories take
special precautions against the accidental introduction of even
a few molecules of a contaminant— especially amplified DNA
from previous experiments. Preventing contamination is a spe-
cial challenge in human applications, such as medicine or the
law, where someone’s life may literally hang in the balance.
Rapid automated PCR has been the key to the extraordinary
upsurge in its applications throughout the life sciences. And the
key to the process’s automation has been Taq polymerase. Taq
is a nickname for Thermus aquaticus, a bacterium that happily
survives and reproduces in an environment that is lethal to
other organisms: hot springs. That is why the organism’s
polymerase is perfectly at home in the rapidly fluctuating
temperatures of automated PCR. Unlike other polymerases, the
enzyme extracted from Taq (and now made in commercial
quantities by genetically engineered bacteria) is stable at high
temperatures. The microbiologists who found these remarkable
organisms decades ago, and then spent years studying their
physiology and biochemistry, had no way of knowing how Fig. 2. A scanning electron micrograph of HIV virus on the surface of T
crucial their work would become to human health, to the lymphocyte (copyright Boehringer Ingelheim International GmbH; photo Len-
forensic sciences, or to the economy. nart Nilsson/Bonner Alba Ab).

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46 POLYMERASE CHAIN REACTION

tion known as otitis media. The technique has detected bacte- tions in body cells. This knowledge will help us take steps to
rial DNA in children’s middle ear fluid, signaling an active prevent those diseases, which are the chief killers in the de-
infection even when culture methods failed to detect it. Lyme veloped world. With PCR analysis of cells shed into feces, for
disease, the painful joint inflammation caused by bacteria example, doctors have already demonstrated premalignant
transmitted through tick bites, is usually diagnosed on the basis changes in the gastrointestinal tract, such as mutations in genes
of symptom patterns. But PCR can zero in on the disease that protect against tumors. This can help them select high-risk
organism’s DNA contained in joint fluid, permitting speedy candidates for colon cancer tests. Researchers have also de-
treatment that can prevent serious complications (Fig. 3). tected potentially metastatic cells in the circulation of patients
PCR is the most sensitive and specific test for Helicobacter with newly diagnosed tumors.
pylori, the disease organism now known to cause almost all PCR can provide enormous peace of mind to people who are
stomach ulcers. Unlike previous tests, PCR can detect three trying to have children—for example, by reassuring anxious
different sexually transmitted disease organisms on a single parents-to-be that they run no risk of having a child with a
swab (herpes, papillomaviruses, and chlamydia) and can even particular genetic disease. The technique even saves the lives
distinguish the particular strain of papillomavirus that predis- of babies before they are born: doctors have used it for exam-
poses to cancer, which other tests cannot do. ining fetal DNA to learn whether the blood groups of mother
In short, if a disorder is caused by an infectious agent, PCR and fetus are incompatible. This condition often leads to severe
can, in principle, ferret out the culprit. More than 60 PCR disability and even death of the fetus, but can be treated
protocols for identifying pathogens have been described to successfully in the womb with enough advance warning—
date, and at least 10 clinical products are available for detecting thanks to PCR (Fig. 4).
the evasive organisms that cause such diseases as tuberculosis, This process is also a direct way of distinguishing among the
chlamydia, viral meningitis, viral hepatitis, AIDS, and cyto- confusion of different mutations in a single gene, each of
megalovirus. which can lead to a disorder such as Duchenne muscular
Because PCR can easily distinguish among the tiny varia- dystrophy. It helps doctors track the presence or absence of
tions in DNA that each of us possesses and that make each of DNA abnormalities characteristic of particular cancers, so that
us genetically unique, the method is also leading to new kinds they can start and stop drug treatments and radiation therapy as
of genetic testing. These tests diagnose not only people with soon as possible. And it promises to greatly improve the
inherited disorders, but also people who carry deleterious vari- genetic matching of donors and recipients for bone marrow
ations, known as mutations, that could be passed to their transplantation.
children. (These carriers are usually not themselves affected by PCR can even diagnose the diseases of the past. Former vice
the mutant gene, which can lead to disease in the next gener- president and presidential candidate Hubert H. Humphrey un-
ation.) derwent tests for bladder cancer in 1967. Although the tests
Research is expected eventually to yield predictive tests: were negative, he died of the disease in 1978. In 1994, re-
methods for finding out who is predisposed to common disor- searchers compared a 1976 tissue sample from his cancer-
ders we do not customarily consider genetic, such as heart ridden bladder with his 1967 urine sample. With the help of
disease, and the cancers that can arise in adulthood via muta- PCR amplification of the small amount of DNA in the 27-year-
old urine, they found identical mutations in the p53 gene,
well-known for suppressing tumors, in both samples. “Hum-
phrey’s examination in 1967 may have revealed the cancerous
growth if the techniques of molecular biology were as well
understood then as they have become,” the researchers said.
Historical medical genetics has gone even further back in
time with PCR. After the color-blind British chemist John
Dalton died in 1844, some tissue from his eyes was preserved.
Dalton had asked for a posthumous investigation of the reason
why he confused scarlet with green and pink with blue. A
recent examination of DNA taken from that tissue, carefully
amplified by PCR, has shown that Dalton lacked a gene for
making one of the three photopigments essential for normal
color vision.
Many of the new genetic tests are the result of the Human
Genome Project, the huge international effort to identify and
study all human genes. Scientists expect the Human Genome
Project to be finished shortly after the turn of the century. It is
moving more rapidly than originally expected toward its ulti-
mate goal, which is to sequence all the DNA in typical human
cells. (“Sequence” means to determine the precise order of the
four different nucleotides that make up any strand of DNA.)
DNA sequencing reveals crucial variations in the nucleo-
tides that constitute genes. These mutational changes produce
disease and even death by forcing the genes to produce abnor-
Fig. 3. mal proteins, or sometimes no proteins at all. DNA sequencing

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POLYMERASE CHAIN REACTION 47

fragments to try to identify individual animals they came

from. The hope is that the genetic information will guide
them in piecing together the 10,000 particles of scrolls that
remain.
PCR is also helping sort out relationships among vanished
human groups, and tracing human migrations. Studies on hu-
man brains that survived 8,000 years in a Florida sinkhole
more or less intact indicate, for example, that the people who
lived there were genetically different from today’s Native
Americans.
Archaeologists are finding that PCR can illuminate human
cultural practices as well as human biology. Analyzing pig-
ments from 4,000-year-old rock paintings in Texas, they found
Fig. 4. The process of amplification by PCR (see Wrobel S. Serendipity, one of the components to be DNA, probably from bison. The
science, and a new hantavirus. FASEB J 9: 1247–1254, 1995). animals did not live near the Pecos River at that time, so the
paleo-artists must have gone to some effort to obtain such an
involves first isolating and duplicating DNA segments for unusual ingredient for their paint. Taking so much trouble
nucleotide analysis. Thus PCR is an essential tool for the suggests that the paintings were not simply decorations, but
Human Genome Project because it can quickly and easily had religious or magical significance.
generate an unlimited amount of any piece of DNA for this PCR can faithfully copy bits of DNA whose age numbers
kind of study. in the thousands—some say millions— of years. Indeed,
PCR’s special strengths may be best revealed in the domain
PCR and the Law that has come to be known as Ancient DNA, where minus-
cule amounts of archaic, badly damaged genetic material are
The technique’s unparallelled ability to identify and copy the the norm. Ancient DNA studies generally fall under the
tiniest amounts of even old and damaged DNA has proved traditional concerns either of archaeology or of evolutionary
exceptionally valuable in the law, especially the criminal law. biology— even the biology of organisms that disappeared
PCR is an indispensable adjunct to forensic DNA typing— long ago.
commonly called DNA fingerprinting (Fig. 5). Scientists have used PCR to correct errors in a previous
To type DNA, for example, DNA extracted from blood analysis of DNA from the 140-year-old skin of the last
found on a murder suspect’s clothes, scientists study a handful quagga, an African member of the horse family. The new
of sites on the DNA where variation among individuals is
typical. This helps them determine the likelihood that the
sample matches the DNA of a specific person, for example a
stabbing victim. Although in its early days DNA typing was
controversial, laboratory standards have been established, and
carefully done DNA typing is now accepted as strong evidence
in courts throughout the world. Defendants’ attorneys continue
to argue about the population frequencies of certain variant
stretches of DNA, but a recent major scientific commentary
concluded, “the DNA fingerprinting controversy has been re-
solved.”
DNA typing is only one of many pieces of evidence that can
lead to a conviction, but it has proved invaluable in demon-
strating innocence. Dozens of such cases have involved people
who have spent years in jail for crimes they did not commit.
One example is Kirk Bloodsworth. The Maryland waterman
was wrongly imprisoned for almost nine years for the rape and
murder of a nine-year-old girl, but was freed in 1993 with the
aid of PCR. Even when evidence such as semen and blood
stains is years old, PCR can make unlimited copies of the tiny
amounts of DNA remaining in the stains for typing, as it did in
Bloodsworth’s case.
“Ancient DNA” and Evolutionary Relationships
Archaeologists have happily seized on PCR and are ap-
plying it in an amazing variety of ways. It is helping, for
example, to launch a new chapter in the colorful and con-
Fig. 5. Polymerase chain reaction (PCR) performed on a Perkin-Elmer thermal
troversial story of the 2000-year-old Dead Sea Scrolls, cycler. PCR enables scientists to copy a single strand of DNA millions of times
which are written on parchment made out of skins from to produce enough DNA for testing (courtesy Perkin-Elmer/William Taufic
goats and gazelles. Researchers are analyzing the parchment Potopgraphy).

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Fig. 6. Illustrations: modification adapted from original, courtesy Perkin-Elmer.

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genetic analysis has shown that the quagga was more closely Crisscrossed with the tiniest of troughs to hold the reagents and
related to the zebra than to any other horse-like creatures. the DNA, the chips are heated electrically and cool down much
By amplifying and analyzing DNA from bone and mummi- faster than the present generation of machines, so amplification
fied soft tissue, scientists have also found that moas, a group is even speedier than today’s swift process. Already research-
of large New Zealand birds that were hunted to extinction, ers have reported using a hand-held battery-powered gadget to
are not related to the still-extant New Zealand kiwi, despite copy pieces of DNA that contained eight different cystic fi-
the fact that both bird species could not fly. Leaping far back brosis mutation sites.
in time, researchers have suggested, however, that termites While such experimental chip-based devices are not yet
imprisoned in amber 40 million years ago differed little ready for prime time, they are hastening the day when scien-
from the termites of today. tists can take them on the road, and patients will be able to get
on-the-spot readouts of their DNA. Before long it may be quite
Modern Systematics, Ecological Studies, and routine to diagnose an infectious or genetic disorder, or even
Animal Behavior detect an inherited predisposition to cancer or heart disease,
But DNA need not be ancient to provide information right in the doctor’s office.
about evolutionary relationships. With PCR, systematists PCR is doing for genetic material what the invention of
can measure differences in DNA sequences between species the printing press did for written material—making copying
directly and, if they select sequences that have changed little easy, inexpensive, and accessible. In principle, PCR can
during evolution, between major classes of organisms. The reproduce the genetic material of any organism in essen-
speed and automation of the process means that scientists tially unlimited quantities, so it can be used to analyze any
can easily compare dozens or even hundreds of individuals, cells containing that material. Whether they are germs, rare
putting their conclusions on a firmer basis (Fig. 6). medicinal plants, or human beings, eventually we can know
With PCR, scientists can glean genetic information from the whatever is recorded in their DNA. With simple organisms,
faintest traces of the shyest, rarest animal— urine, feces, scent to know their DNA will be to know almost everything about
marks, infinitesimal bits of hair or skin rubbed onto a tree—as them. With complicated ones, like people, DNA is only part
the elusive creature passes by. In addition to information that of the story, but a very big part. Thanks to PCR, we will be
aids classification, individuals can be identified so as to esti- probing the genetic past, and peering into the genetic future,
mate population size in a particular locale, or to determine the for many years to come.
geographic range of a single animal, or a group of them. The
technique can be adapted to similar studies of plants, for SUGGESTED READING
analysis of patterns of seed dispersal and the relative repro-
ductive success of specific plants. Researchers have even used Kary Mullis tells how the idea for PCR came to him out of
PCR to study badly damaged specimens such as roadkill, or the the blue in “The Unusual Origin of the Polymerase Chain
leavings of carnivores, where little-known vertebrates have Reaction,” Scientific American, April 1990, p. 56 – 65.
been identified among the prey. The medical applications of PCR change almost daily. A. F.
Because PCR does not require invasive samples of blood Markham reviewed many of them in “The Polymerase Chain
or other tissue, research need not disrupt an animal’s life- Reaction: A Tool for Molecular Medicine,” British Medical
style—a boon for behavioral studies—and should not dis- Journal 306: 441– 447, February 13, 1993. PCR applications
tress people concerned about animals. DNA extracted from are also explored in a recent series of short articles on molec-
feces, for example, is being explored to find out which of the ular medicine appearing in The New England Journal of Med-
approaches to mating common among olive baboons work icine. See especially two articles by Stephen P. Naber: “Mo-
best, by establishing which males actually are successful at lecular Pathology: Diagnosis of Infectious Disease” (331:
fathering infants. 1212–1215, November 3, 1994) and “Molecular Pathology:
Researchers have used the technique to aid in reducing Detection of Neoplasia” (331: 1508 –1510, December 1, 1994.)
illegal trade in endangered species, and products made from See also “Human DNA Polymorphism” by David Housman
them. Because PCR is a relatively low-cost and portable tech- (332: 318 –320, February 2, 1995) and “Molecular Diagnosis”
nology, and likely to become more so, it is adaptable for field by Bruce Korf (332: 1499 –1502, June 1, 1995).
studies of all kinds in the developing countries. It is also a tool For everything you ever wanted to know about PCR and the
for monitoring the release of genetically engineered organisms
field of genetics, consult the second edition of Recombinant
into the environment.
DNA, by James D. Watson, Michael Gilman, Jan Witkowski,
and Mark Zoller (Scientific American Books, 1992). It contains
THE FUTURE OF PCR
an entire chapter on PCR, and discussions of many of the
The present technology for doing PCR, about the size of a technique’s applications in genetics are sprinkled throughout.
microwave oven and costing several thousand dollars, seems An immense amount has been written about the forensic
destined for further radical improvement. By tinkering with uses (and misuses) of DNA analysis via PCR; some of it has
variables such as chemical reagents and pH, researchers have probably appeared in your local newspaper. For an authorita-
already reported success at copying larger and larger pieces of tive overview, see Genetic Witness: Forensic Uses of DNA
DNA, including the entire genome of HIV. Tests, a substantial report by the Office of Technology Assess-
Extraordinary miniaturization of the hardware is also under- ment of the US Congress. (US Government Printing Office,
way, as experimenters squeeze PCR onto chip-sized devices. OTA-BA-438, July 1990). For a recent slant that emphasizes

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50 POLYMERASE CHAIN REACTION

the complications of PCR as a forensic tool, see Jon Cohen, ronmental science, and ecology were reviewed by Norman
“Genes and Behavior Make an Appearance in the O. J. Trial,” Arnheim, Tom White, and William E. Rainey in “Application
Science 268: 22–23, April 7, 1995. of PCR: Organismal and Population Biology,” BioScience 4:
“Ancient DNA” was described by its leading authority, Svante 174 –182, March 1990.
Pääbo, in Scientific American, November 1993, p. 87–92. Survey PCR’s future technology with Robert F. Service,
The virtually unlimited uses of PCR in evolutionary biology, who described “The Incredible Shrinking Laboratory,” in Sci-
zoology, botany, animal behavior, conservation biology, envi- ence 268: 26 –27, April 7, 1995.

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