Polymerase Chain Reaction

Polymerase Chain Reaction

MARK V. BLOOM, PhD

DNA LEARNING CENTER

Cold Spring Harbor Laboratory

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, 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 Corp. 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 from 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 he 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 a single-strand 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. 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 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; 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 C, during which the DNA is denatured into single strands;
2. one to several minutes at 50-65 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 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 aquanticus , which inhabits hot springs. This enzyme, called Taq polymerase, remains active despite repeated heating during many cycles of amplification. Second, DNA thermal cyclers were invented that uses 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 modification 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 genetics 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 amplifications, 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 amplification 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 micrograms) 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 in the same gel. Since the amplified sequence represents a relatively large proportion of the lambda genome (48,052 base pairs), the efficiency of the reaction is increased, and only two temperatures are required for each replication cycle. Primer annealing and extension occur together as the reaction incubates at the lower temperature and rises to the denaturation temperature of the next cycle.

Automated DNA thermal cyclers are becoming less expensive, and may soon cost about the same as micro centrifuges, 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 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 lysed by boiling and centrifuged to remove the 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 afford 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.