Polymerase Chain Reaction

Mark V. Bloom, Ph.D.
DNA Learning Center,
Cold Spring Harbor Laboratory,
Cold Spring Harbor, New York 11724

Louis Pasteur once remarked that "chance favors the prepared mind," and certainly the history of scientific progress supports his contention. The annals of science provide numerous examples of serendipitous discovery. Some are mythic, such as Newton's discovery of gravity following his encounter with an apple; while others are more rooted in fact - like Fleming's discovery of penicillin on a contaminated petri dish.

Scientists today continue to take unexpected turns on their paths to discovery. One such recent detour occurred in 1983 on U.S. Route 101 in northern California. Kary Mullis (Fig. 1), a scientist working for the Cetus Corporation, was driving along the mountain road with a friend one moonlit night. His mind constantly shifted from the road to a problem of nucleic acid biochemistry. He was struggling to devise a simple method for determining the identity of a specific nucleotide along a stretch of DNA. It seemed that just as he solved one technical problem, another one took its place. Suddenly, a flash of insight caused him to pull the car off the road and stop. He awakened his friend dozing in the passenger seat and excitedly explained to her that he had hit upon a solution - not to his original problem, but to one of even greater significance. Kary Mullis had just conceived of a simple method for producing virtually unlimited copies of a specific DNA sequence in a test tube - the polymerase chain reaction (PCR).

The polymerase chain reaction was introduced to the scientific community at a conference in October 1985. Scientists, quick to embrace the new technique, were surprised (with the wisdom that accompanies hindsight) that no one had thought of it earlier. Cetus rewarded Kary Mullis with a $10,000 bonus for his invention, and later, during a corporate reorganization, sold the patent for the PCR process to the pharmaceutical company Hoffmann-La Roche for $300 million! The popularity of PCR continues unabated. As of the end of 1993, PCR has been referenced in well over 7,000 scientific publications.

DNA Hybridization

The chemistry of PCR, as with much of molecular biology, depends on the complementarity of the DNA bases. When a molecule of DNA is sufficiently heated, the hydrogen bonds holding together the double helix are disrupted and the molecule unzips or "denatures" into single strands. If the DNA solution is allowed to cool, then complementary base pairs can reform (renature) and the original double helix is restored.

Experiments performed during the 1960s demonstrated that many DNA sequences were not unique within the genome. Purified DNA solutions were denatured by heat and then allowed to cool. Using a spectrophotometer it was possible to monitor the rate at which the DNA renatured. Data from these studies revealed that genomes are composed of different classes of DNA sequences that can be distinguished by their repetitive frequency. For example, some amphibian cells contain more DNA than human cells, owing to a large excess of repetitive DNA relative to their human counterparts.

While useful for studying the broad outline of genome organization, this approach could not be used to investigate the structure of individual genes. This ability came about during the 1970s following the introduction of DNA restriction analysis and nucleic acid hybridization techniques. Hybridization allows a specific DNA sequence to be analyzed against the complex background of a eukaryotic genome. It is estimated that the human genome contains between 100,000 and 200,000 genes. To focus on an individual gene, DNA from the target organism is isolated, fragmented with restriction enzymes, and separated by gel electrophoresis. The DNA fragments are denatured to render them single stranded and exposed to a solution containing a radioactive DNA "probe." The probe consists of single-stranded nucleic acid (either DNA or RNA) with a sequence chosen to base pair with the gene of interest. Under appropriate conditions of temperature, salt, and pH, called "stringency," the probe will bind to its corresponding sequence in the target DNA and nowhere else. The presence of a radioactive signal (often by exposure to X-ray film) indicates positions of probe binding.

The Mechanism of PCR

The polymerase chain reaction is a test tube system for DNA replication that allows a "target" DNA sequence to be selectively amplified, or enriched, several million-fold in just a few hours (Fig. 2). Within a dividing cell, DNA replication involves a series of enzyme-mediated reactions, whose end result is a faithful copy of the entire genome. Within a test tube, PCR uses just one indispensable enzyme - DNA polymerase - to amplify a specific fraction of the genome.

During cellular DNA replication, enzymes first unwind and denature the DNA double helix into single strands. Then, RNA polymerase synthesizes a short stretch of RNA complementary to one of the DNA strands at the start site of replication. This DNA/RNA heteroduplex acts as a "priming site" for the attachment of the DNA polymerase, which then produces the complementary DNA strand. During PCR, high temperature is used to separate the DNA molecules into single strands, and synthetic sequences of single-stranded DNA (20-30 nucleotides) serve as primers. Two different primer sequences are used to bracket the target region to be amplified. One primer is complementary to one DNA strand at the beginning of the target region; a second primer is complementary to a sequence on the opposite DNA strand at the end of the target region.

To perform a PCR reaction, a small quantity of the target DNA is added to a test tube with a buffered solution containing DNA polymerase, oligonucleotide primers, the four deoxynucleotide building blocks of DNA, and the cofactor MgCl2. The PCR mixture is taken through replication cycles consisting of:

  1. one to several minutes at 94-96 degrees C, during which the DNA is denatured into single strands;
  2. one to several minutes at 50-65 degrees C, during which the primers hybridize or "anneal" (by way of hydrogen bonds) to their complementary sequences on either side of the target sequence; and
  3. one to several minutes at 72 degrees C, during which the polymerase binds and extends a complementary DNA strand from each primer.

As amplification proceeds, the DNA sequence between the primers doubles after each cycle. Following thirty such cycles, a theoretical amplification factor of one billion is attained.

Two important innovations were responsible for automating PCR. First, a heat-stable DNA polymerase was isolated from the bacterium Thermus aquaticus, which inhabits hot springs. This enzyme, called the Taq polymerase, remains active despite repeated heating during many cycles of amplification. Second, DNA thermal cyclers were invented that use a computer to control the repetitive temperature changes required for PCR.

Following amplification, the PCR products are usually loaded into wells of an agarose gel and electrophoresed. Since PCR amplifications can generate microgram quantities of product, amplified fragments can be visualized easily following staining with a chemical stain such as ethidium bromide. While such amplifications are impressive, the important point to remember is that the amplification is selective - only the DNA sequence located between the primers is amplified exponentially. The rest of the DNA in the genome is not amplified and remains invisible in the gel.

Applications of PCR

Following the introduction of PCR, the technique spread through the community of molecular biologists like - well, a chain reaction. As more scientists became familiar with PCR, they introduced modifications of their own and put the technique to new uses. Almost overnight, PCR became a standard research technique and the practical applications soon followed. Not surprisingly, the first applications to leave the laboratory dealt with detection of genetic mutations.

PCR has proven a quick, reliable method for detecting all manner of mutations associated with genetic disease - from insertions, to deletions, to point mutations. Some enthusiasts predict that within five years most genetic testing will be PCR-based.

Duchenne muscular dystrophy is an example of a genetic disease whose detection has been greatly simplified by the use of PCR. The human dystrophin gene, spread out over two million base pairs of DNA on the X chromosome, is the largest gene identified to date.

Boys afflicted with Duchenne muscular dystrophy have deletions in the protein coding regions (exons) of the dystrophin gene. The gene's great size makes it impractical to examine its entire length for mutations, so a technique called "multiplex PCR" is used to sample various regions of the gene from one end to the other. The technique involves simultaneous amplification from nine different sets of primers, all within the same test tube. Each set of primers is chosen to produce a different-sized amplification product from a different region of the dystrophin gene. Following amplification, the PCR products are analyzed by gel electrophoresis. Boys having a normal dystrophin gene will display nine different-sized amplification products, while boys with deletions in the gene will be missing one or more of the PCR products.

PCR can also be used to detect the presence of unwanted genetic material, as in the case of a bacterial or viral infection. Conventional tests that involve the culture of microorganisms or use of antibodies can take weeks to complete or be tedious to perform. PCR offers a fast and simple alternative. For example, in the diagnosis of AIDS, PCR can be used to detect the small percentage of cells infected by the human immunodeficiency virus (HIV). DNA isolated from peripheral blood cells is added to a PCR reaction containing primers complementary to DNA sequences specific to HIV. Following amplification and gel electrophoresis, the presence of an appropriate-sized PCR product indicates the presence of HIV sequence and therefore, HIV infection.

The sensitivity of PCR is so great that signals may be obtained from degraded DNA samples and sometimes from individual cells. This ability and the inherent stability of DNA have combined to permit DNA to be amplified from some unusual sources, such as an extinct mammal called the quaga, an Egyptian mummy, and a three-million-year-old termite trapped in amber. This situation has, almost overnight, transformed ignored museum collections of biological specimens into treasure troves of genetic information. Evolutionary biologists are using these specimens and PCR to explore the genetic relatedness of organisms across species boundaries and now even across time.

When PCR is used with degraded DNA samples, it can synthesize an amplification product, even if the sample's average fragment size is less than the distance between the primer binding sites. During PCR, overlapping fragments within the target sequence can function as primers to generate full-length amplifica-tion products. This ability of PCR to utilize degraded DNA samples is of great interest to forensic scientists who must sometimes work with human cells in very poor condition. The technique has provided conclusive identifications in cases where conventional DNA typing has failed. Ironically, the greatest concern about the widespread use of PCR in forensic medicine is the technique's extreme sensitivity. Even miniscule amounts of DNA left over from previous amplifications can be reamplified, leading to an inconclusive result.

PCR in the Classroom

Educators who wish to introduce PCR to their students can do so by designing an experiment that doesn't push the limits of the technique. Most importantly, one should use target DNA from an organism with a small genome.

Lambda DNA provides a simple and inexpensive source of target DNA. Almost any region of the lambda genome can be selected for amplification. Since PCR amplifies small fragments more efficiently than larger ones, choose primer sequences to yield an amplification product of between 500 and 1,000 base pairs. Start with one or two nanograms (0.001 microgram) of target DNA. This is a lot of DNA by PCR standards, but is still undetectable (or just detectable) in ethidium bromide stained gels. Following just 10 PCR cycles, a theoretical amplification of 1,000-fold is achieved - sufficient to visualize the PCR product in the gel. By amplifying duplicate samples for increasing numbers of cycles, a time course of the amplification can be seen by electrophoresing the samples together in the same gel. Since the amplified sequence represents a relatively large proportion of the lambda genome (48,502 base pairs), the efficiency of the reaction is increased, and only two temperatures are required for each replication cycle (Fig. 3). Primer annealing and extension occur together as the reaction incubates at the lower temperature and raises to the denaturation temperature of the next cycle.

Automated DNA thermal cyclers are becoming less expensive, and may soon cost about the same as microcentrifuges, which are increasingly found in teaching laboratories. A DNA thermal cycler enables the instructor to take full advantage of PCR's capabilities, including the amplification of human DNA. PCR can allow students to analyze their own DNA and see for themselves their unique genetic heritage.

The 100,000 or more genes found in the human genome constitute perhaps 5% of the approximately 3.5 billion base pairs of DNA sequence in the haploid human genome. Most of this noncoding DNA lies between genes and has been called spacer, or even "junk" DNA. Although the biological significance of this DNA is still a matter of conjecture, some noncoding sequences have proven useful in the diagnosis of genetic disease and in paternity/forensic determinations. Of particular interest are families of repeated DNA sequences, where copies of a single repeated unit are linked in tandem, one after the other. The number of repeats can vary from one allele to another and, therefore, from one individual to another. Alleles with varying numbers of repeats can be separated by size using gel electrophoresis. Such repeated sequences are termed VNTRs, for variable number of tandem repeats. VNTRs are especially useful in demonstrating the polymorphic nature of human DNA and provide the basis for paternity/forensic analysis of DNA samples.

To perform a human DNA fingerprinting experiment, students obtain a sample of their own cheek cells using a saline mouthwash (bloodless and noninvasive). The cells are collected by centrifugation and resuspended with a resin, which binds metal ions that inhibit the PCR reaction. The cells are lyzed by boiling and centrifuged to remove cell debris. A sample of the supernatant containing chromosomal DNA is added to a test tube holding the PCR reaction components, placed into a DNA thermal cycler, and taken through 30 cycles of amplification.

The primers used in the experiment bracket a VNTR locus and selectively amplify only that region of the genome. Following amplification, student alleles are separated according to size using agarose gel electrophoresis. After staining with ethidium bromide, one or two bands are visible in each student lane - indicating whether an individual is homozygous or heterozygous for that VNTR locus. Different alleles appear as distinct bands, each composed of several billion copies of the amplified allele. A band's position in the gel indicates the size (and number of repeat units) of the VNTR allele; smaller alleles move a longer distance from the origin (sample wells), while larger alleles move a shorter distance.

There is consensus among educators that students best learn science by doing it. Unfortunately, as science becomes increasingly dependent on high technology, it becomes more difficult to give students access to the latest tools of science. The power and simplicity of PCR affords the teacher a rare opportunity to give students "hands on" experience with a still evolving technology on the leading edge of genetic research. While such lab experience may not stimulate every student's interest in science, it can help to increase the number of "prepared minds" able to exploit opportunities of chance.

Further Reading

Allen, R., et al. 1991. Analysis of the VNTR locus AmpliFLP D1S80 by the PCR followed by high resolution PAGE. American Journal of Human Genetics 48:137.

Chamberlain, J.S., et al. 1990. Multiplex PCR for the diagnosis of Duchenne muscular dystrophy. In PCR Protocols: A Guide to Methods and Applications (M.A. Innis, et al., eds.). Academic Press, New York, pp. 272-281.

Micklos, D.A. and G.A. Freyer. 1990. DNA Science: A First Course in Recombinant DNA Technology. Carolina Biological Supply Company, Burlington, NC and Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

Mullis, K.B. 1990. The unusual origin of the polymerase chain reaction. Scientific American 262:56-65.

Paabo, S., R.G. Higuchi, and A.C. Wilson. 1989. Ancient DNA and the polymerase chain reaction. Journal of Biological Chemistry 264:9709-9712.

Saiki, R.K., et al. 1985. Enzymatic amplification of beta-globin sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230:1350-1354.

Equipment Requirements for Agarose Electrophoresis

Ray Gladden
Biotechnology Department

One of the most common questions asked by customers is "What type of equipment do I need to get started?" Equipment is required for the following manipulations: micropipetting, electrophoresis, and visualization.

Micropipetting, or dispensing microvolumes of DNA, is best done by using adjustable micropipettors with disposable tips. These are relatively expensive, ranging from $200-$300 each depending on the brand purchased. If these are out of your budget range, a good inexpensive alternative is the Wiretrol microcapillary pipet, with a disposable tip and a reusable plunger. This option is not only inexpensive but is also very accurate and fairly easy to use. For dispensing premeasured volumes of predigested DNA, use disposable plastic needlepoint pipets.

Electrophoresis is the separation of DNA fragments into different sizes through a matrix of agarose or acrylamide inside an electrophoresis chamber. The chamber has two electrodes on opposite ends, which can be attached to a DC power supply. Gel chambers are available with single (J8-21-3668) and double (J8-21-3654) casting trays. The double casting tray enables you to run two gels at one time in the same gel box.

An electrophoresis buffer fills the chamber and conducts the electricity between two electrodes. When current is applied, the negatively charged DNA migrates toward the positive electrode. The agarose gel acts as a sieve, allowing the smaller-sized fragments to migrate faster than the larger fragments, thus separating fragments by size.

The speed of electrophoresis is dependent on the size of the gel and the amount of voltage applied to the gel box by the power supply. The higher the voltage, the faster the migration of the fragments. Each gel box has a maximum optimal voltage range, and exceeding this range results in smearing of the DNA bands. Lower voltages generally give cleaner separation of bands. We offer several power supplies (see pages 18-19), allowing you considerable flexibility in performance and cost.

After electrophoresis, view the DNA by staining with ethidium bromide, methylene blue, or Carolina Blue(TM) DNA stain. Ethidium bromide is the stain most commonly used by researchers because of its sensitivity to DNA and the speed of staining. Drawbacks include the cost involved for its visualization (it requires a UV light source), and its suspected carcinogenicity. If you wear rubber gloves and work in a sink, the low concentrations required for staining can be used safely.

Methylene blue stain is less sensitive than ethidium bromide, but this can be compensated for by using higher concentrations of DNA. It is also a visible-light stain, which means UV light sources are not required. Many instructors purchase inexpensive white light boxes, or utilize an overhead projector protected by a sheet of acetate. Another method is to place a clear piece of acetate over the gel and trace the DNA band patterns. A disadvantage of methylene blue is the time required for staining and destaining the gel. An alternative to these two stains is the Carolina BLU(TM) staining system.

Staining Agarose Gels with Carolina BLU(TM) DNA Stain

Shirle Pace
Biotechnology Department

Students active in DNA science are eager to visualize the bands of DNA migrating through the agarose gels. Developed in cooperation with Dr. Greg Freyer of Columbia University, Carolina BLU(TM) stain has the ability to stain DNA during electrophoresis, allowing for early visualization of the bands. With its more intense staining and easier visualization of smaller bands of DNA, which are not usually seen after the destaining of methylene blue, now many gels are ready for visualization immediately after electrophoresis. This allows for faster analysis and interpretation of the results.

This is accomplished by adding a small amount of a concentrated solution of the stain to the gel and buffer; the stain is picked up by the DNA as it migrates through the agarose gel. During electrophoresis, the positively charged stain particles in the agarose migrate towards the negative electrode of the gel box, thus the necessity for adding stain to the buffer. As the DNA passes the midpoint of the gel in its migration towards the anode, without the replacement stain from the buffer, it will actually lose stain in the clear portion of the gel.

After electrophoresis, additional staining of the gel with a dilute solution of Carolina BLU(TM) stain in a staining tray intensifies all the bands with short staining (15-20 minutes) and short destaining (1 hour or less) times. Easy agitation of the gel in the tray facilitates staining and destaining. Distilled or de-ionized water should always be used for destaining because tap water contains chloride ions that remove stains from DNA and agarose within a few hours. Changing the water a few times speeds removal of the stain for easy visualization in less than an hour. Gels can be stored in a few milliliters of water in a covered stain tray for several weeks and even refrigerated in an airtight bag for a few months.

For optimum performance, consider the following tips. Less DNA is required in a digest of lambda DNA than if you were using methylene blue. Specific amounts of lambda and plasmid DNA are listed in the package instructions. Because temperatures above 50degrees C cause the stain to fade to ineffectiveness, melted agarose is best cooled until the container is comfortable to the touch before adding the appropriate microliters of Carolina BLU(TM) stain. Follow the instructions exactly for adding stain to the gel and buffer because extra stain can cause artificially precipitated DNA bands in the gel, which detracts from the usual banding pattern. Strictly following stain amounts for your voltage requirements provides excellent results in gels, which can be stored for future viewing and analysis.

The final stain can be reused eight times, and buffers can be reused four or five times unless stain was added. Buffer with stain added can be reused one time in the same day, but it fades and loses its effectiveness if stored longer. Gels and buffer may be prepared one day early for lab the next day. Gels may be photographed with the same cameras, white lights, and yellow filters used for methylene blue photography. Safe and easy cleanups are another advantage of Carolina BLU(TM) stain.

Gene Future

Thomas Lee. 1993. 339 pages. The author brings together science, politics, economics, and our legal system in this far-reaching study of the promise and dangers of biotechnology. He cites detailed examples of already existing transgenic plants and animals, nearly accomplished cures of genetic diseases, legal cases involving DNA fingerprinting, and much more. For middle school and up. Hard cover.
   J8-45-2457B     Each . . $24.95

Book Watch

From The Book Doctor

The Future Is Now

DNA fingerprinting. The origin of life and of our own species. Genetic testing and gene therapy. Transgenic tomatoes and designer babies. From the courtroom to the medical research center, from farming to pharming - today's headlines proclaim that the biotechnology revolution impacts on us all.

Certainly, everyone who wants to make informed decisions must attempt to understand the essentials of DNA science and genetics. If, like me, you are enthralled by the complexity of modern science but usually feel as if you're an outsider peering through a translucent window, then Thomas Lee's Gene Future: The Promise and Perils of the New Biology (J8-45-2457B) will be a welcome addition to your reading.

Warning: Once you begin reading Gene Future, or Lee's earlier success, The Human Genome Project, you're likely to get hooked. If you're a science researcher, teacher, or student, then okay, you're improving your professional status. But if you're just an interested dilettante like me, who's fascinated by the way our 100,000 or so genes control our lives, then watch out!

What is Gene Future about? A few of the author's headings provide a flavor: Genes, Genealogy, and Ancient DNA; The Garden of Eden; Animal Enhancement Engineering; and Food - or "Frankenfood"? But don't let the puns and allusions mislead you. This is serious science without being scholarly, educational without being pedantic. While Gene Future does not deliver the technical detail of superior textbooks like James Watson's Recombinant DNA, or Paul Berg and Maxine Singer's Dealing with Genes: The Language of Heredity, Professor Lee does provide an overview of the structure and coding of DNA, how the polymerase chain reaction works, the mechanisms of gene therapy, and many other related processes.

In fact, most of Lee's book concentrates on the practical applications of genetic engineering. As the pace of the Human Genome Project escalates, scientists are finding more reasons to believe they will be able to successfully manipulate fundamental life processes at the molecular level. Instead of treating the symptoms of disease, doctors will modify our genes to remove the cause. This will eventually mean the control and elimination of genetic disorders like cystic fibrosis and muscular dystrophy. Gene therapy will also be used to prevent many kinds of cancer and heart disease, as well as to direct the body's own immune system to fight off threats of all kinds.

But the new biology brings perils as well as pearls. As Lee concludes in Gene Future: "We will have the opportunity to diagnose, treat, and prevent diseases that have brought suffering into the lives of so many. In so doing we will collect the most intimate data about our personal genetic endowments. This information may be used to safeguard our health and that of future generations. This record of our genes also could be misused unless we can ensure that access to it is limited to legitimate purposes. . . . We can only hope that our human intelligence and creativity. . . will be supported by the wisdom to make the choices that will work for the common good of all life."

Carolina BLU(TM) DNA Stain

Reduces staining and destaining time!

An ultra-safe, two-step staining system in which a small amount of stain is added to the agarose and buffer prior to running the gel. This causes DNA bands to become faintly visible during electro-phoresis. After electrophoresis, simply stain as usual to enhance visualization of bands.

Carolina BLU(TM) stain gives better results than methylene blue because of increased sensitivity, reduced background, reduced staining time (stains in 15-20 min), and reduced de-staining time. Includes 30 mL of stain to be added to the agarose and buffer solutions, and 250 mL of a final staining solution.

   J8-21-7300     Per set . . $4.95

Polymerase Chain Reaction Kits

A revolution in molecular genetic analysis brings the power of PCR* into your classroom!

Since its development by Kary Mullis and coworkers in the mid-1980s, the Nobel-Prize-winning technique of polymerase chain reaction (PCR)* has revolutionized molecular genetic analysis. Now, under exclusive license from the Perkin Elmer Corporation, Carolina Biological offers the first PCR kits designed for the teaching laboratory. Both kits were developed in cooperation with the DNA Learning Center of Cold Spring Harbor Laboratory.

Our Introductory Kits allow students to efficiently amplify lambda DNA in only 30 minutes, using either two water baths or a Perkin Elmer DNA thermal cycler, and analyze results on agarose gels.

Our Advanced Kit offers a safe, practical means to generate personal DNA fingerprints.

Your students learn cutting-edge technology with PCR kits from Perkin Elmer and Carolina Biological!

Polymerase Chain Reaction is covered by patents owned by Hoffmann-La Roche.

DNA Amplification by Polymerase Chain Reaction

Our introductory kits use PCR to amplify a 1,106 base-pair sequence from the bacteriophage lambda genome. A sample of dilute lambda DNA is mixed with a "cocktail" of amplification reagents and amplified manually using two water baths (55degrees C and 100degrees C), or by using a Perkin Elmer DNA thermal cycler. Following amplification, samples are loaded in a 1% agarose gel, electrophoresed, and stained with ethidium bromide or methylene blue. The size of the expected amplification product is verified by comparison to DNA size markers. Materials are sufficient for 50 reactions or 12-16 time-course experiments. The Amplification Reagents Kit includes lambda DNA, PCR mix, magnesium chloride solution, mineral oil, DNA size markers, and teacher and student instructions. The Amplification and Electrophoresis Reagents Kits includes all of the above, plus 0.5-mL PCR tubes, 1.5-mL tubes, electrophoresis buffer, loading dye, staining solution (either ethidium bromide or methylene blue), agarose, staining trays, and disposable gloves. Major equipment needed but not included: 55degrees C and boiling water baths (or a DNA thermal cycler available from Perkin Elmer, 850 Lincoln Centre Drive, Foster City, CA 94404), gel electrophoresis chamber, power supply, and micropipetor. Specify use date to ensure fresh materials. Shipped 2nd day on wet ice.