Oligos To Go! : Purveyors of Custom Oligos

Date: March 30, 1998 Author: Jim Kling Table of Vendors So--the boss calls you into her office and tells you it's time to find another supplier of oligonucleotides. The custom house you've been using just went out of business, or maybe your team is preparing to delve into a new research project that calls for oligos. You scan the ads in several journals and do a quick internet search. Before you know it you've got a 'short' list of 50-100 suppliers. Wading through a sea of suppliers to find the right fit is a daunting task, but LabConsumer is here to help. We spoke to a number of researchers whose uses of oligos include DNA sequencing, DNA diagnostics, and developmental and clinical antisense research (in which DNA or RNA sequences that are complementary to a coding sequence are introduced--they hybridize to messenger RNA, preventing it from taking part in protein synthesis). We also spoke to suppliers and quality control specialists. Here's what they told us. Since every research application places unique demands on reagents, "ask other people in [your] field," says Charles Roth, an instructor in bioengineering at Massachusetts General Hospital (Boston, Mass). "Ask who they use, how they decided on them, how pure is the stuff, how quickly [they] get it." Of the numerous attributes that one should consider when ordering from a custom oligo house--including cost, purity, time to delivery, and customer support--Roth's primary concern is purity, since the success of his preclinical studies depends on reducing the variables introduced by impure reagents. "An antisense experiment is a lot more stringent than if you're using primers in a molecular biology application. Both the effective therapeutic dose and the presence of non-specific effects are influenced by the oligo purity," he says. Most custom houses will do the purification for you, but it can be an expensive service,Roth points out. A custom house's standard purification may be done using gel electrophoresis or size exclusion columns, but Roth has used reversed-phase chromatography to purify samples before use, although he notes that others use ion exchange and capillary electrophoresis to do the job. Following synthesis, the final oligo product contains a hydrophobic protecting group at the 5' end, slowing the oligo's passage through the column while impurities--including shortened oligos that failed a coupling step along the way--pass through quickly. To be dead certain of your material, Roth suggests taking it one step further: Digest the oligo into its individual bases and analyze its composition on a precalibrated column. "My feeling is if you want to be sure of [the oligo's purity], check it out yourself." On the other hand, many basic research applications are more forgiving. "For sequence finishing and for PCR [polymerase chain reaction] work, you can be fairly cavalier--we don't even purify or desalt [the oligos we make]," says Harold Garner, Assistant Director of the Genome Sequencing Center at the University of Texas Southwestern Medical Center (Dallas, Texas). But Garner echoes Roth's sentiments when it comes to other research his group is doing. "Oligos for things like DNA chips and hybrid assays have to be extremely high in purity." The single biggest complicating factor, says Garner, is the efficiency of the coupling process. The standard phosphoramidite chemistry used in oligo synthesis has an efficiency of 99 per cent, meaning that in every cycle only 99 per cent of the growing chains actually add the next base. An unpurified sample of an oligo made up of 60 -- 70 bases will contain only a small fraction of the full-length sequence. "In some cases that is very important, in particular with DNA chips, because chips are oftentimes used to detect single base pair mismatches," Garner points out. For-profit custom synthesis houses are not the only option available to researchers. Many universities and research institutes support "core facilities," which, in addition to doing peptide synthesis, DNA and RNA sequencing, and various other services, also do custom oligo synthesis. Originally set up to pool funds when reagents and synthesis processes were more expensive (and less reliable), core facilities aim to meet the needs of an organization's researchers at prices competitive with those of for-profit custom synthesis houses. In fact, 3-4 years ago they could easily beat commercial prices, but a large increase in the number of for-profit suppliers-driven by advances in synthesis technology and simplification of instrumental procedures--brought on a price war that resulted in prices similar to those of core facilities. "The core lab is run to provide full service, [including] consultation and planning strategy," says Gregory Buck, chair of the Association of Biomolecular Research Facilities (ABRF www.medstv.uni-melb.edu.au/abrf.html) Nucleic Acids Research Committee, director of the Nucleic Acid core laboratory at Virginia Commonwealth University (Richmond, Va), and co-owner of Richmond-based custom house Commonwealth Biotechnologies, Inc. "Most custom synthesis houses don't provide that service. The cost of just making oligos has dropped significantly [in part] because companies basically are just turning out oligos at very high rate without any auxiliary services or consultation," Buck adds. Most agree that core facilities on average do just as good a job of producing oligos as custom houses. Roth, who has purchased oligos from a number of different custom houses over the years, recently discovered the core facility at Massachusetts General Hospital. "The oligos they make are as good or better than the ones we order outside of the system." A typical custom house may churn out thousands of oligos per day, while core facilities are usually not equipped to handle such a large volume. The experienced researcher who knows what his or her needs are--and therefore can order in larger volumes and likely receive a discount from a custom house--will probably be better served by a commercial facility, says Buck. But for those unfamiliar with the lay of the oligonucleotide landscape, advice and support from core facility staff may be a welcome addition. "If a person needs [advice] on how to interpret results, how to [figure out] what went wrong with an experiment, or how to design an oligo, a core lab can help out much more than a commercial company," says Buck. Core labs can also respond much faster: whereas a commercial housemay take 3 days to fill a request, Buck's own facility can often deliver an oligo in 4-6 hours. Not surprisingly, customers of core facilities come primarily from their own institutions, though ABRF maintains a list of facilities that will supply outside researchers. "[But] the advantage of working with a core lab is that the guy is down the hall and you can [interact directly] with him; if the guy is across town or at some other locale, that defeats the purpose," Buck points out. Even so, "the core director might be more willing to discuss the ins and outs of a project than a commercial house would," he adds. If you are considering a core lab's services but want to check on its quality, the good news is that ABRF's Nucleic Acids Research Committee runs a program to check up on core facilities, testing their output for accuracy and reproducibility. Not every core facility participates in the program, but those who do receive feedback on how they're doing (upon which they presumably act) and hence may be more reliable sources. The committee publishes its results annually as a research article in the journal Biotechniques, in a newsletter. Okay, finding suppliers that can deliver a vanilla oligo of reasonable purity for a reasonable price is all well and good, but suppose you're looking for an oligo with radio-labels, tags, or specialized linkers incorporated. Then what? The good news is that most commercial houses will handle these sorts of orders, according to Chris Boles, Director of Research at Mosaic Technologies, Inc. (Boston, Mass). "I would say that the large names in the custom synthesis industry are pretty good quality, and they will handle modifications found in most suppliers' catalogs," says Boles, whose company is developing DNA diagnostics and a novel method for solid-phase PCR. "But they'll charge you for the whole bottle [of labeling reagent], so it's a good idea to order the oligos in bulk," says Boles. Some linkers or tags can be trickier than others. "We ordered five modified oligos from one commercial house, and none [of the lots] contained more than 5 per cent of modified oligos. We ordered the same thing from another house and the lots came back around 95 per cent modified When you're using special modifications, it's really important to ask the house how many they do a year. After the thing completely failed we asked the first house and they said they did one a year [previously]. The other place had done 60 in the previous 3 months," Boles recalls. Researchers in need of really specialized or unusual modifications have one other option--synthesis consultants. "Most of the commercial oligonucleotide houses offer very basic services," says Richard Hogrefe, president of Trilink Biotechnologies, Inc. (San Diego, Calif). "We are a group of research scientists who came out of the therapeutic and diagnostic fields to sell modified compounds," says Hogrefe. He says many of Trilink's customers are referred to them by custom houses that realize they can't satisfy the customer's needs. Of course, core facilities can also provide specialized service, but Hogrefe maintains that his service is a step above: "I'm a classically trained oligochemist, with published research in designing and synthesizing oligos. [Core labs] have a lot of information to offer, but they won't be able push frontiers for you," he says. Another oft-considered question among oligo users is when to buy the synthesis equipment, roll up the sleeves, and produce the oligos in-house. Buying an instrument is of course an expensive proposition--maybe even more so than people realize. Buck has witnessed a number of faculty splurging on their own instruments. "Usually [the instruments] sit idle for long periods of time--then when they are used they have to be reconditioned. If you're using 10 oligos a week it doesn't make sense," he maintains. Garner has produced an instrument (Science, 276:1489, 1997) capable of producing 192 oligos a day, nearly quadrupling the 48-oligo capacity of the ABI machines (Perkin-Elmer Corp., Norwalk, Conn) that are most commonly used. Garner is making the information and services available to anyone, including plans, software, and manufacturing. He estimates the total cost of manufacturing and assembling to be about $65,000. In evaluating whether to build his instrument or to buy a commercial product, "[you] just have to do the math," says Garner. "Once you make the capital investment in machines and ancillary equipment and hire someone to maintain them, you probably need another $50,000 to do quality control--so you're looking at a $100-$150,000 capital investment, plus hiring someone to maintain it. If you use 1 megabase of oligos per year at [a production cost of] 10 cents a base, versus 65 cents a base to purchase them at a custom house, that's a savings of 50 cents a base--or $500,000 a year." However, people planning on going this route should be aware that according to Applied Biosystems, a Division of The Perkin-Elmer Corporation and the holder of numerous patents covering oligoncleotide synthesis, neither Garner nor the University of Texas are licensed to the technology. Under a licensing program, Applied Biosystems makes available the rights to practice oligonucleotide synthesis. Whatever sources you use, chances are good that just one oligo supplier may not be the best answer to all of your needs. Mosaic uses several, according to Boles. "Some houses really specialize in exotic dyes and modifications. We use one house that specializes in custom dyes for one out of every five orders. For unmodified PCR primers, we use a very low-priced discount house. Because we do a lot of work with fancy DNA probes, we have a custom chemistry company that we deal with for very specialized applications." If you visit http://www.ispex.ca/nacccustomprod.html. you'll find links to web pages for custom production companies (not just oligos). Jim Kling is a writer and consultant based in Bellingham, Wash. He can be reached at jkling@nasw.com. Table of Vendors

Oligos To Go! : Purveyors of Custom Oligos

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