Molecular biologists and geneticists depend on the ability of DNA synthesizers to produce man-made oligonucleotides (oligos) of defined sequence for a variety of genetic studies, from the isolation of genes not clonable by other techniques to the diagnosis of mutations responsible for human genetic diseases. Research facilities specializing in recombinant DNA techniques typically own or have access to high-throughput DNA synthesizers, which are capable of simultaneously manufacturing up to 15 micromoles (one millionth of a mole) of four independently programmed DNA or RNA sequences.
Individual laboratories, however, do not need the full range of capabilities of these high-throughput instruments, and often cannot afford their cost. As a result, these labs have traditionally relied on the services of a central DNA synthesis facility or have purchased oligos on a custom basis from commercial suppliers.
But in recent years a new class of low-cost, high-speed DNA synthesizers has been developed. These new devices, capable of producing up to 15 micromoles of highly purified oligonucleotide sequences, have made it possible for smaller laboratories to synthesize man-made genes.
In 1968, Har Goribind Khorana and his colleagues at the University of Wisconsin, Madison, were the first to succeed in chemically synthesizing a polynucleotide, the transfer RNA (tRNA) for alanine. Although this was indeed a historic achievement - one that won Khorana the Nobel Prize in Physiology and Medicine - there were no known biological applications for the technique at the time. As a result, the synthesis of genetic material remained in the domain of organic chemists, who refined Khorana's solution phase methods and applied his techniques to synthesize DNA and other nucleotide polymers.
But the synthesis of genes remained a difficult process until the advent of recombinant DNA techniques in the 1980s created a demand for synthetic DNA, which motivated chemists to improve the process, increase yields and sequence lengths, and automate the procedure.
One of the most important advances in the manufacture of artificial oligos involved the introduction of solid-phase synthesis, a rapid, automated method for assembling amino acids developed in 1963 by Robert Bruce Merrifield of Rockefeller University, New York. Merrifield was awarded the Nobel Prize in Chemistry in 1984 for developing this revolutionary technique, which DNA chemists applied to oligo synthesis. In this method, the first oligo is anchored on a solid support, such as glass or silica gel, and then each additional nucleotide is added in the desired order to make the oligonucleotide chain. Armed with this advance and other improvements in synthesizing techniques, manufacturers developed high-throughput devices and, more recently, the compact, easy-to-operate DNA synthesizers for the small molecular biology lab.
"Five years ago, DNA synthesis was in the hands of experts at core research facilities, who understood the complex chemistry and machinery of early-model synthesizers," says Timothy McGrath, marketing manager at Milli-Gen/Biosearch, Burlington, Mass. "But since manufacturers have simplified the chemistry, improved the hardware, and prepackaged reagents and nucleotide bases, DNA synthesis has become a tool for the nonspecialist user who needs synthetic DNA but doesn't need to know the chemical details," he says.
DNA synthesizers, in essence, are fluid-handling devices. They automatically control the flow of various reagents and nucleotides into a reaction vessel, where the internucleotide phosphate bonds are formed to create the desired oligonucleotide chain. Since DNA monomers contain several chemical groups that can react with the reagents during synthesis, the nucleotides are equipped with protective chemical groups at various sites to eliminate the likelihood that side reactions will occur. Part of the synthetic process involves removing these protective groups at designated stages during synthesis. Most protective groups remain permanently attached to the oligonucleotide chain throughout the synthesis and are removed after chain assembly. Others are needed to ensure the proper positioning of the next monomer, and therefore can be removed after each elongation cycle.
The most common synthetic method, phosphoramidite chemistry, involves a series of deprotection, coupling, capping, and oxidation steps that are repeated until the specified nucleotide chain is constructed. The sequence begins with a protected monomer chemically bound to the inorganic matrix of the glass support. The support, contained in the reaction vessel, interacts with reagents released into the chamber at specific times throughout the cycle. Between each step, the column is washed with solvent to remove excess reagents, but the support with the growing oligonucleotide chain remains intact.
The initial step of the synthesis (deprotection) involves an acidic wash, which removes the protective group on the terminal nucleotide on the chain. The next nucleotide in the sequence is activated and added to the vessel, where it forms an internucleotide phosphite-triester linkage with the terminal nucleotide. This new coupling is then oxidized to the more stable phosphotriester linkage, and any unreacted hydroxyl groups are acetylated by the subsequent addition of acetic anhydride. This acetylation, or capping step, renders the site on the growing chain inert and facilitates purification of the final oligo chain. After this four-step cycle is repeated for each added nucleotide, treatment with ammonia cleaves the oligonucleotide chain from its support, and an extended heating period removes all protective groups from the oligonucleotide chain.
To facilitate the production of oligomers that are cell permeable and resistant to cellular degradation, some units have software for running hydrogen-phosphonate synthesis chemistry. The overall operation of this method is very similar to the phosophoramidite method, except the oxidation step occurs after construction of the chain is completed because a more stable coupling reagent is used. This method enables researchers to modify the entire phosphate backbone with sulfur or other chemical groups.
Since users are more interested in DNA production than in the chemical methodology used, a built-in microprocessor or stand-alone computer automatically controls the delivery of reagents to the reaction vessel, using preprogrammed or user-defined protocols. Researchers need only key in the desired oligonucleotide sequence, load the reagents into position, and initiate the synthesis.
Applications Compact DNA synthesizers are becoming popular among small microbiology labs. The ability to easily synthesize specific DNA sequences within the lab instead of contracting out for these services is a major advantage. In the neurology department at the University of Pennsylvania School of Medicine in Philadelphia, five researchers share one of the small DNA synthesizers from MilliGen/Biosearch for a broad range of recombinant DNA applications.
Although these researchers had purchased oligos from a nearby synthesis facility for $3 a base, they find it is much more convenient to have the unit in the lab. "Instead of waiting three to four days' turnaround time for our oligos from the core facility, we can have our sequences in one day," says Larry Wrabetz, one of the postdoctoral fellows that has access to the device. While he says the cost savings are minimal - only $0.50 a base - the ability to synthesize genes as needed is a definite plus.
Some core synthesis facilities have also purchased these small units as adjuncts to their high-output devices. William Wunner, director of the DNA synthesis facility at the Wistar Institute, Philadelphia, produces more than 150 oligonucleotide chains each month. While this synthesis center operates two high-throughput devices, the researchers also use one of the smaller units to synthesize oligonucleotide chains with modified bases, in response to special requests.
"When researchers request sulfonate oligonucleotides at every phosphate linkage, we can't dedicate one of the high-output devices to handle these requests. It would interrupt the flow," says Timothy Horsley, one of the research technicians at Wunner's lab. "The smaller unit fills this need."
Synthetic DNA has a variety of uses in the molecular biology laboratory. The majority of applications of synthetic oligonucleotides are in the area of recombinant DNA. Researchers routinely insert chemically synthesized gene fragments of a desired sequence into naturally occurring or hybrid genes to isolate and alter biologically important genetic sequences using other recombinant DNA techniques. They also use man-made genes as probes to identify DNA sequences that encode proteins for which only partial amino acid sequences are known. Some other uses include producing defined sequences of oligonucleotides as primers for gene amplification by the polymerase chain reaction (PCR) technique, isolating genes not clonable by other techniques, and preparing genes and modified genes for proteins having potential clinical applications.
Several companies manufacture various DNA synthesizers. Which device to choose depends on each individual lab's production and experimental needs.
- The PS 250A from Cruachem, Herndon, Va., produces DNA sequences of 0.2 to 1.0 micromoles up to 250 bases long using phosphoramidite chemistry. The cycle time is 10 minutes, and couplings are said to be more than 98% efficient. Menu-driven software runs on an IBM PC PS-2 model 30 or 50 computer. Software for RNA synthesis and to accommodate synthesis up to 10 micromoles will be available later this year.
- The 391 PCR-MATE ER DNA Synthesizer from Applied Biosystems Inc., Foster City, Calif., produces sequences of RNA or DNA up to 10 micromoles that are up to 249 bases long using phosphoramidite or hydrogen-phosphonate chemistries. The cycle time is 5.6 minutes, and couplings are said to be more than 98% efficient. Menu-driven software runs on the built-in microprocessor, which interfaces with a PC for downloading sequences and printouts.
- The DNA synthesizers available from Biotix Inc., Danbury, Conn., sequence DNA strands up to 200 bases long using phosphoramidite or hydrogen phosphonate chemistries. The cycle time is 3 to 5 minutes, and couplings are said to be 98% to 99.9% efficient. Menu-driven software runs on an IBM-compatible PC with 640K RAM. Sequences up to 150 bases can be programmed. The DNA 200 synthesizer is optimized for 0.05 micromole synthesis scale, while the DNA 101 and DNA 102 produce up to 0.5-, 1.0-, and 10-micromole samples. In-line detritylation monitor provides an automatic evaluation of the efficacy of each step of the synthesis; in-line flow monitor warns of improper flow.
- The Synostate D device from B. Braun Diessel Biotech Inc., Allentown, Pa., will be available later this year. Sequences of DNA or RNA up to 200 bases long can be synthesized using phosphoramidite chemistry. The menu-driven software runs on a built-in microprocessor, but an RS-232 interface links the unit to a PC. Self-diagnostics program and sensors warn of improper syntheses.
- The Gene Assembler Plus from Pharmacia LKB Biotechnology, Piscataway, N.J., sequences 0.2-, 1.3-, and 10-micromole samples of DNA up to 186 bases long using phosphoramidite chemistry. The cycle time is 7.5 minutes (17.5 minutes for 10-micromole sequences), and couplings are said to be more than 98% efficient. Programmable, menu-driven software runs on IBM compatible PC. Printer is optional.
- The Generator from E.I. Du Pont de Nemours Co., Wilmington, Del., produces sequences of DNA of 0.25 or 1.0 micromoles, up to 100 bases long using phosphoramidite chemistry. The cycle time is less than 8.5 minutes. Menu-driven software runs on a built-in microprocessor. Parallel printer interface is available.
- The Cyclone Plus DNA Synthesizer from MilliGen/Biosearch, Burlington, Mass., produces 0.2-, 1.0-, or 15-micromole sequences of DNA or RNA up to 200 bases long using phosphoramidite or hydrogen phosphonate chemistries. The cycle time is 5 to 6 minutes, and the coupling efficiency is said to be 99.7% to 99.9%. Programmable, menu-driven software runs on a built-in microprocessor. Parallel printer interface is available.
- The Model 300 DNA synthesizer from Vega Biotechnologies, Tucson, makes 0.25-, 1-, or 5-micromole sequences of DNA up to 190 bases long using phosphoramidite chemistry. The cycle time is 7 minutes per column, and the coupling efficiency is 98.4%. Menu-driven software runs on a built-in microprocessor and 5-inch CRT screen.
Carole F. Gan is a freelance science writer based in Philadelphia.