Device Helps Find DNA 'Needles' In Genetic 'Haystack'

In recent years, the clinical diagnosis of genetic and infectious dis- eases has relied on the analysis of specific DNA nucleotide sequences. And for molecular biolo gists, the task of finding a particular target strand, only a minute fraction of the total DNA, has been a burdensome challenge—like searching for a needle in a haystack. Now, however, GeneAmp PCR, a product based on proprietary technology of Cetus Corp., Emeryville, Calif., not only helps scientists find that one needle in the haystack, it also allows them to make millions of copies of it—in effect creating a whole new stack of only target needles, thus simplifying the study of the previously tiny traces of the DNA. The technology has broad applications in research into genetic disease such as sickle cell anemia and cystic fibrosis, into genetic predisposition to diseases such as diabetes, and into somatic mutations involved in the development of cancer. It also is useful for forensic analysis of tissue samples such as hair and bloodstains, and for tissue typing associated with organ transplantation and paternity determination. By simplifying and shortening the process for analyzing gene sequences, this gene amplification technique—automated two years ago and recently improved—is fueling rapid advances in molecular biology research. The new technique, known as polymerase chain reaction, or PCR, is a an in vitro method for making millions of copies of minute amounts of a specific target DNA sequence in only a few hours. PCR amplification uses two oligigonucleotide primers. The scientist creates these primers, short pieces of DNA whose sequences have been synthesized to complement the DNA in the target region, to define the ends of DNA that he or she wants to duplicate. Then the DNA sample is heated to separate the strands, and each oligonucleotide primer binds to one of the two separated DNA strands. Because the priming step provides obligate start sites for replication to begin, the DNA sequence in the area bounded by the primers is copied. This step, carried out by the enzyme DNA polymerase, yields two new strands complementary to the original two. Millions Of Copies At each subsequent cycle of heating, cooling, and synthesizing, the amount of target DNA is doubled. Millions of copies can be produced in a few hours, making further detection and analysis easy. The cycles in the PCR procedure require these periods of heating and cooling to denature and synthesize the DNA. Originally, PCR was performed using a DNA polymerase from the bacterium E. coli, which is inactivated at high temperatures, thus making it necessary to add new enzyme at each cycle— a tedious and expensive procedure. But scientists at Cetus, where the process was developed, found that DNA polymerase from a bacterium that lives in hot springs and geysers, Thermus aquaticus (Taq), is capable of surviving undamaged through the heating part of the PCR cycle. This solves the problem of replenishing enzymes at every cycle and also increases the specificity of the amplification reaction. Synthesizing the new DNA strand at higher temperatures prevents the accumulation of nonspecifically primed amplification products and ensures that the specific target sequence is the predominant product. In the August 5,1988, issue of the Proceedings of the National Academy of Sciences (page 5698), scientists at the University of California, Los Angeles, the Howard Hughes Medical Institute, Children’s Hospital at Stanford, and Cetus, reported using the PCR technique for detecting and differentiating between chronic myeloid and acute lymphocytic leukemia by diagnosing the molecular presence of the Philadelphia chromosome. With GeneAmp PCR, clinicians could eventually make diagnoses that would take weeks with other methods of chromosome analysis. In addition, the standard cytological test for this disorder requires that between 1% and 5% of cells harbor the altered chromosome; PCR can detect one altered cell in a million normal cells. Also, in the August 5 issue of Science (page 708), cell biologists at the University of California, San Francisco, reported on a new technique based on Cetus’ PCR. Scientists extracted RNA from a few macrophages found in a mouse wound. This RNA was converted to DNA and multiplied several billion times using PCR technology The scientists were then able to identify tiny amounts of genetic messengers produced by single cells in the body. They were able to show that in an actual wound— rather than in cell culture—the scavenger cells of the macrophages produce several growth factors to stimulate healing. Last month, the New England Journal of Medicine (September 1, 1988, p. 537), carried a report by Cetus scientists in collaboration with a team at Johns Hopkins University that described how the use of PCR can significantly simplify genetic diagnosis procedures. In this study, prenatal diagnosis for sickle-cell anemia and beta-thalassemia was carried out in a simple four-hour test (known as a “dot blot”) using nonradioactive oligonucleotide probes. This same procedure can be used for other genetic diseases as well as for genetic typing for individuality in forensics and paternity determination. In June 1988, scientists at Northwestern University Medical School and at Cetus showed that with the PCR procedure they can detect the presence of AIDS viral DNA in individuals who had not yet produced antibodies to the virus. They also demonstrated the ability of the procedure to detect the virus during latency, when antigen tests are ineffective, which has value in diagnosis and prognosis, and also in monitoring the success of therapeutic treatment. Cetus and Eastman Kodak Co. have licensed two California reference laboratories to use PCR technology for detecting the genes of the AIDS virus. The procedure, HIV DNA by PCR, is available through Specialty Laboratories Inc., based in Santa Monica, and Pathology Institute, based in Berkeley, and can be ordered by a physician directly from one of the laboratories or through a hospital or commercial laboratory. Through a joint venture with Perkin-Elmer Corp., Cetus has introduced the DNA Thermal Cycler gene amplification machine and the GeneAmp reagent kit. The thermal cycler, priced at $8,500, is a microprocessor-controlled temperature cycling instrument designed to perform the rapid temperature changes and incubations necessary for PCR gene amplification. The GeneAmp research reagent kit ($395) provides Taq as well as other reagents used in PCR, enough to perform 100 assays at amplification of at least 100,000-fold after 25 standard cycles. Since PCR is capable of producing millions of copies from a single progenitor sequence, it represents a kind of “cell-free molecular cloning.” This powerful technique is already finding broad application in all areas of molecular biology research and promises to play a pivotal role in the development of DNA-based diagnostic tests for genetic and infectious diseases. Henry A. Erlich is director of human genetics at Cetus Corp., Emeryville, Calif.

Device Helps Find DNA 'Needles' In Genetic 'Haystack'

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