Innovative Alternatives To PCR Technology Are Proliferating

Since the earliest days of the biotechnology endeavor, scientists have sought a practical way of co-opting the complex biological machinery that regulates DNA. From the moment they identified the intriguing enzymes that choreograph the dance of DNA replication, biotechnologists began developing ways to commander them; the swivelases and gyrases, forms of unwinding proteins that untwist the molecule; the nucleases, which snip it; the strands; the ligases, which tie these strands together; and even a contingent of repair of repair enzymes, which oversees the entire, multistep event. The polymerase chain reaction (PCR) capitalizes on this biological toolbox by manufacturing millions of copies of desired piece of DNA. Invented in 1985 by Kary Mullis, Henry Erlich, and their colleagues at Cetus Corp. of Emeryville, Calif. (Science, 230: 1350-4), the technique has had an enormous impact on the field of biotechnology. Cetus joined with the Norwalk, Conn.-based Perkin-Elmer Corp. to form Perkin-Elmer Cetus Instruments, which markets PCR. The process has been patented by Perkin-Elmer Cetus, but other companies are quickly developing alternative methods of replicating DNA, expanding the approach and its possibilities even further. The polymerase chain reaction rapidly replicates a selected piece of DNA in test tube. The recipe includes a target DNA sequence; two synthetic, single-stranded oligonucleotide primers that are complementary to opposite ends of the target sequence; a hefty supply of the four types of DNA nucleotide building blocks; and most important , Taq1, a DNA polymerase produced by Thermus aquaticus, a microbe that inhabits deep-sea thermal vents. This heat-stabile polymerase is an adaption to its host's naturally hot environment; and it is the key to the success of the technique. When PCR debuted, heat labile polymerase from Escherichia coli was used, necessitating the addition of more polymerase each time the temperature was raised. Taq1 renders this unnecessary, streamlining and simplifying the previously cumbersome technique. PCR consists of three cycling stages. First, heat is applied to denature (separate) the two strands of the target DNA. The second step introduces specificity-temperature is lowered, and the two short DNA primers are added. They anneal to denatured target DNA by complementary base pairing. In the third step, Taq1 inserts the appropriate bases onto the primers, following the sequence specifications of the target DNA. The new strands serve as templates in the next round of replication, which begins immediately. The pieces of DNA accumulate geometrically, so that after just 20 cycles, 1 million copies of the original sequence float in the test tube. PCR can replicate DNA of 100 to 10,000 bases. Because of its broad applicability, PCR is edging its way into a potpourri of fields, from archaeology to zoology. It revealed the tragic fate of a couple's missing son, for example, by amplifying DNA in a smear of brain tissue on the bumper of an abandoned car, and provided a workable sample of DNA from another brain, that of a 7,000-year-old mummy. The technique also speeds a variety of diagnostic tests for inherited or infectious diseases. DNA is a direct calling card of an infection, and amplifying that DNA is quicker than waiting for an immune response to reach detectable levels. PCR's greatest strength is that it works on crude samples of rare and minute sequences. Its greatest weakness, oddly enough, is its exquisite sensitivity. If, for example, a diagnostic sample is contaminated by a leftover bit of DNA from a previous run, or a stray hair or eyelash from the technician conducting the procedure, a false positive can result-a particularly distressing complication in a clinical situation. Even a bit of viral DNA lodged in a chromosome from a past infection can botch the process, causing the Taq1 polymerase to faithfully replicate an incorrect or altered sequence. Uses of PCR extend beyond the faithful replication of a specific sequence. In sited-directed mutagenesis, the primers are synthesized to differ slightly in sequence from the corresponding regions of the target DNA. The result-copies bearing the difference, like typesetting an original from a manuscript, and perpetuating the change in copies of the document. Researchers use this approach to stitch cutting sites for particular restriction enzyme into a gene. When the amplified DNA is then inserted into a cloning vector, it can be clipped out by virtue of its custom-designed restriction sites. A technique called inverse PCR turns the tables, allowing DNA on either side of a target sequence, rather than the sequence between the primers, to be amplified. This method is used by gene mappers trekking through genomes, and to identify regulatory elements that flank genes. PCR's limiting requirement of having a known target sequence can be overcome for highly conserved sequences, those genes are very similar in base-pair lineup between species. Typically, primers are synthesized from a known mouse gene and are then used to bracket a human counterpart of that gene. A mouse gene, for instance, recently led researchers to the gene behind the devastating human disease neurofibromatosis. Since PCR techniques have been patented by Perkin-Elmer Cetus, efforts to commercialize a competing system can lead to legal problems, others seeking to exploit natural DNA replication are turning to different enzymes. And the future looks bright: Several industry analysts forecast a $1 billion worldwide market for nucleic acid-based diagnostic tests by 1995. As yet, none of these PCR alternatives is commercially available, however. A rundown of the front-runners follows: Q-beta Replicase This amplification system, based on an enzyme called replicase, from the RNA virus Q-beta, is quite different in concept from PCR and, its developers hope, will compete with PCR on the clinical front because of its comparative simplicity. "We acquired the Q-beta replicase system from Columbia University and the Salk Institute, and received exclusive worldwide licensure in 1988," says Jim Richards, director of business development at Gene-Trak Systems in Framingham, Mass. The Q-beta replicase system does not amplify a target DNA sequence, but mass-produces an RNA complementary to a particular DNA sequence (Bio/Technology, 6:1197-1202, Oct. 1988). It consists of a hybrid RNA built of a sequence called MDV-1 from Plasmodium faciparum, a parasite of protozoans, plus a short sequence of RNA that functions as a "reporter" because it is complementary to the DNA sequence of interest. The MDV-1 RNA is a natural template for the replicase. When mixed with the sample DNA, the engineered RNA seeks its DNA mate and, in binding, contorts to activate the replicase, prompting it to copy the MDV-1 and its tag-along RNA sequence. As in PCR, each product RNA serves as a template for the next round of replication. Within a half-hour, a billion copies of RNA are produced. They are detected by ethidium bromide staining. The kinetics of the Q-beta replicase reaction offer information on the amount of target DNA in a sample. As product accumulates, the enzyme becomes linear, rather than geometric, to the logarithm of the original amount of RNA. For example, if sample A stains three times as intensely as sample B, it contains a 1,000 times as much target DNA as sample B. The ability to calibrate amounts, and the constant temperature of the reaction (37 C), make the Q-beta replicase system "elegantly suited" for diagnosis, says Gene-Trak Systems' Richards. "For diagnostics using probes, Q-beta is the best, in terms of cost, ease of use, and less sophisticated instrumentation," he adds. "But for research, PCR and some of the others may be better." The system at first was sharply criticized for a high false-positive rate, due to enzyme instability and unfaithful replication of non-target sequences, but, according to Richards, the glitches have now been worked out. Ligase Chain Reaction The ligase chain reaction (LCR), being commercialized by Biotechnica International Inc.'s diagnostics branch, in Cambridge, Mass., is perhaps the most straightforward of the amplification techniques. Prefabricated abutting segments of a target sequence plus a thermostabiler ligase are added to a sample. The segments bind to their complementary sequence (viral DNA, for example), serve as additional targets for the next round.As in PCR, millions of copies are present after 20 to 30 cycles. But the elegant LCR lacks the broadness of PCR. "The LCR is inherently more limited than PCR because in PCR, if you know something about the region, you can get at the sequence and into those regions you don't know about yet," says Keith Backman. senior vice president for technical affairs at Bio-Technica, referring to inverse PCR's power to nap genes. "LCR is small, on the order of 50 base pairs, and assumes you don't know the sequence already. There is no way to learn something new, so it is more limited to diagnostics-to questions of what's there." But what it lacks in flexibility, LCR makes up in fidelity: In LCR, unlike PCR or Q-beta replicase, the reaction halts if the target sequence is not present. A hybrid gene amplification scheme involving both a polymerase and a ligase is in the works at Im-Clone Systems Inc., located in New York. The company's proprietary repair chain reaction (RCR) is "highly specific in works nicely compared to PCR," says David Segev, head scientist for the project. The technique is expected to be ready for commercialization in two to three years, he adds. Nucleic Acid Sequence-Based Amplification Because of their simplicity, Q-beta replicase and LCR are well suited for the medical marketplace. Nucleic acid sequence-based amplification, on the other hand may, remain in the research lab because of its complexity. This technique uses several enzymes and an intricate cycling pattern of RNA and DNA synthesis. Basically, cDNAs (made using reverse transcritse from DNA sequence serve as templates, guiding the buildup of the desired sequence. This method is extremely sensitive, claim its developers at Cangene Corp., based in Mississauga, Ontario. It is able t spot and amplify as few as 10 molecules among 100 billion others. With 10 to 10(4) molecules of starting template, it can produce .5 to 5 micrograms of RNA. A reaction runs for about an hour and a half, and all templates, enzymes, and primers are added at the start, says Larry Malek, group leader at Cangene. Signal Amplification Biochemicals other than nucleic acids can be harnessed to herald a target DNA sequence's presence. While PCR allows a researcher to detect a sequence by generating a measurable amount of DNA, signal amplification works by more than amplifying the DNA sequence itself, these systems amplify a chemical that binds to the DNA, thereby signaling its presence. This makes them especially useful for diagnostic purposes in which large amounts of the actual DNA are not required. The hub of the AmpliProbe systems is bacteriophage M13, which carries both the sequence of interest (the primary probe) and binding sites for a series of secondary probes. Using this system, a researcher can detect 0.8 picogram of hepatitis B DNA against a background of 100 nanograms of human genomic DNA. "AmpliProbe's sensitivity can't compete with PCR, because it is limited by the rate of signal generation," says ImClone's Jian Qing Yang, group leader for AmpliProbe. But this system is very fast-in total it takes six hours." Chiron Corp. in Helsinki, Finland; and Digene Diagnostics Inc. of Silver Spring, Md., are also developing signal amplification systems. Profile Diagnostics Sciences Inc., located in New York, offers a new twist on detecting a specific DNA sequence in a sample. Invented by founder and president Chai-Gee Wang, the "homogenous gene assay" is run in a cuvette, where DNA probes are bound to a solid support. The single-stranded partial probes are added. "The bound probe might be 25 bases, the single-stranded partial probe, 15 bases," says Wang. The trick is that each partial probe is bound to a microbead harboring either a fluorescent molecule or heavy metal atom. Added sample DNA hybridizes with greater affinity to the anchored, 25-base probe than to the 15-base partial probe. As a result, the partial probes are displaced from their microbeads and enter the surrounding solution, where their "reporter" components are detected by light or X-ray fluorescence. Wang says his approach is faster and "cleaner" than PCR. Another plus is that different probes can be used in a single cuvette, making it possible to detect more than one DNA sequence per sample. This could be valuable, for example, in the diagnosis of opportunistic infections due to AIDS, or in tracking hormone levels. The invention of the polymerase chain reaction in 1985 was, to be sure, a great leap forward in the rapidly changing field of molecular biology. Now, alternative gene amplification techniques, with strengths that complement the original, are expected to push the limits of biotechnology even further in 1990s. It will be interesting to watch these alternative systems carve their niches in the years to come. Ricki Lewis teaches biology at the State University of New York, Albany. She is the author of The Beginnings of Life (William C. Brown, Dubuque, Iowa, 1991).

Innovative Alternatives To PCR Technology Are Proliferating

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