Special Needs: Custom Houses that Provide Specialized Oligos

LabConsumer recently took a look at custom oligo houses (J. Kling, The Scientist, 12(7) 18, March 30, 1998), and found that there's more than one kind of company: those that produce standard (commonly referred to as vanilla) oligos quickly and, for the most, part cheaply and those that specialize in oligos with an ever-increasing array of tags and modifications. Since there wasn't the time or space to do the latter group of companies justice in the previous article, this article revisits custom oligos, focusing on them. But first is a brief compendium of some of the special features currently in use: Nuclease resistance: Antisense oligos are finding a place in both therapeutic and research arenas. To enhance their efficacy, modifications to oligos have been introduced to make them resistant to nuclease digestion. A commonly used modification for this is the substitution of phosphodiester bonds with phosphorothioate bonds, either throughout the length of the oligo or only in part. Partially modified oligos--so-called chimeras or "gap-mers"--have modified 3' and 5' ends with a normal phosphodiester core, which makes the ends resistant to exonuclease activity and makes the oligo less toxic to cells. Alternatively, chimeric oligos with 2'-O-Methyl RNA can be made that also have enhanced nuclease resistance and have increased binding affinity. Fluorescent tags: Also useful in antisense applications are fluorescent tags, which can be used to track the intracellular location and to determine the life of the oligos once they have been taken up by cells. A variety of fluorescent tags are available: fluorescein dyes, rhodamine dyes, BODIPY, and Cy3 or 5 dyes. Oligos bearing fluorescent tags are also used in automated DNA sequencing, fluorescent in situ hybridization, PCR product detection, and a variety of DNA fragment analysis protocols. Modified bases: Incorporating modified bases can make duplexes with oligos more stable and can facilitate the preparation of degeneracy in primer sequences. Any one of a number of modified bases can be used, such as deoxyinosine and uridine. Fluorogenic dye quencher probes are the hottest and latest addition to the field. Originally introduced for quantitative PCR, these oligos and their cousins, molecular beacons, are increasingly finding uses in other applications. These oligos are constructed with a fluorescence dye in proximity to a quenching molecule and fluoresce only when quenching is released through some event, such as displacement and nuclease activity by Taq polymerase during PCR. RNA oligos and analogs: RNA oligos made with modified bases have been found to have higher affinity for DNA, without the inherent nuclease sensitivity and instability of RNA. Tags: A variety of other tags can be put onto oligos to aid in the capture of the molecules (biotinylated oligos, for example, can be recovered using streptavidin-coated media); to make them reactive (adding a primary amine makes oligos receptive to other labels); to permit ligation to other nucleic acids (5´-end phosphates do this); or to determine kinetic properties, as in the case of radioactive labels. Peptide nucleic acids: This new class of informational molecule has a neutral "peptide-like" backbone with the same nucleobases attached as in DNA. The unique properties of these molecules impart a number of abilities: to penetrate into the chromosome, to resist degradation of any kind, and to hybridize with higher affinity and specificity than corresponding oligonucleotides. PE Applied Biosystems PE offers primers and probes for PCR, TaqMan and sequence detection, GeneScan, linkage mapping and custom sequencing applications. The Custom services offered are: ABI PRISM® fluorescent labeled primers for use with the ABI PRISM® DNA 377 and 310 Sequencers. The primer sequences and choice of labels are user defined. The fluorescent dyes available for 5' labeling are 6-FAM, HEX, TET, NED, TAMRA and ROX. Synthesis is offered in three sizes, the minimum quantity being 4,000 pmoles. However for human linkage studies, primer sequences and labels are provided that have been optimized for best performance in mapping. These markers are provided in whole sets, panels covering each chromosome and as individual loci. ABI TaqMan™ probes are available in three synthesis sizes; the minimum quantity is 5,000 pmole. Sequence detection primers for use with these probes in assays are also provided in complimentary sizes. In partnership with Hybridon (Worcester MA) scales of synthesis from 0.5 gram to several kilos of DNA are available. With the new merger of Perkin Elmer and PerSeptive Biosystems, a full range of PNA oligos and reagents are offered. PE Applied Biosystems offers several phosphoramidites for on-instrument labeling that enable the addition of labels, linkers for coupling other labels, and the means to add a phosphate during oligonucleotide synthesis. 800-762-4000 www.perkin-elmer.com Cruachem Cruachem's custom DNA and RNA synthesis service offers a comprehensive list of 104 modifications for DNA and RNA oligonucleotides, including 5 ´ -biotinylation, 5 ´ -fluorescein, triple DNP labeling, and S-oligos at up to 1 mmole scale. The company is actively involved in the research and development of second generation anti- sense oligos and labels to satisfy ever-evolving needs. Oligos are supplied desalted or at different levels of purification to meet specific project requirements: PAGE for oligos above 50 bases and for modified oligos; HPLC for >95% purity of modified oligos; and COP for 90-95% purity, suitable for most applications. Cruachem's custom RNA service uses two different chemistries, and with targeted ion exchange, can supply RNA oligos at >95% purity. In addition, ribozymes, dual labeled RNAs and RNA/DNA chimeras are available. The finished product can be shipped with the protecting groups on or off at the request of the customer and can be HPLC purified (up to 30 bases) or PAGE purified (30-40 bases). Orders are accepted on a dedicated fax line and processed with a management system that includes a number of checks throughout the process to minimize errors. 800-327-9362 www.cruachem.com Dharmacon Research, Inc. Dharmacon Research has been developing a novel 2´-acid labile chemistry for RNA synthesis originally invented at the University of Colorado at Boulder. Dharmacon's custom RNA synthesis service is now providing the research community with a reliable source of RNA oligonucleotides, in advance of introducing the RNA synthesis reagents and methodology. Currently, RNA oligonucleotides up to 36 bases long and scales up to 1 µmole are available, at a cost of $9.50 per base for 0.2 µmole scale. Dharmacon Research reports that yields are generally two to four times more than that obtained with other RNA chemistries and that a clean major product is routinely observed, often not requiring further purification. Dharmacon Research is continuing to develop this technology and to introduce modified bases and backbones. 800-235-9880 Integrated DNA Technologies IDT makes specialized modifications for oligonucleotides for use in antisense studies, both phosphorothioate-substituted oligos and 2 ´ -O-Methyl RNA oligos. HPLC purification is recommended for all oligos used in antisense studies to ensure both chemical purity and the removal of truncated, short oligos that arise during synthesis. For phosphorothioates, 1 µmole scale reactions usually produce 30-100 OD260 units of DNA. After HPLC purification, a minimum yield of 20 OD260 units is guaranteed. Ten µ-mole scale reactions produce 300-500 OD260 units of DNA and a minimum yield of 200 OD260 units of DNA after HPLC is guaranteed. 800-328-2661 www.idtdna.com Macromolecular Express DNA EXPRESS at Macromolecular Resources (Fort Collins, Colo.) uses the latest technology to synthesize, purify, and qualify every oligo. All oligos are quality controlled by MALDI-TOF mass spectrometer and/or gel electrophoresis. Modifications that can be incorporated into oligonucleotides include biotinylation ($50), phosphorylation ($40), fluorescein ($50), Texas Red ($100), rhodamine ($100), digoxigenin ($150), FAM ($150), HEX ($150), amine ($30), phosphorothioated oligos ($25 first bond, $2.50 each additional bond), and numerous minor bases. All modification prices are in addition to the per base price of the different synthesis scales starting at $1 per base. RNA synthesis is also available starting at $20 per base for the 0.2 mmole desalted scale. All oligos come with a full warranty. 800-491-0424 http://mmr.bmb.colostate.edu/dna/mod.html Monomer Sciences, Inc. Monomer Sciences designs novel nucleosides based on 20 years of experience in nucleoside chemistry: base labile 5´ or 3´ hydroxyl-protecting groups, linkers and supports, rare nucleosides, and 2 ´ modifications. Monomer Sciences also offers contract research and product development under the strictest confidentiality. Available nucleosides include standard protecting groups as well as rare items. The catalog lists nucleoside products according to their protection state--fully protected to bare. The Custom Manufacturing Services division provides ultra-pure products produced under cGMP guidelines. 800-4DNARNA www.monomer.com Oligos Etc. Oligos Etc. is a GMP facility offering a range of specialized services in the custom synthesis of DNA and RNA oligonucleotides. The company offers a full list of tags and modifications, including fluorescent tags, conjugates, 3' and 5' amino modifiers, thiol modifiers, and minor nucleotides. The company also offers antisense oligomers, including p-ethoxies, phosphorothioates, chimeric oligos, and oligos with methyl-phosphonate linkages. Oligos Etc. recently introduced optimized antisense oligomers with fewer phosphorothioate linkages, increased hybrid affinity, and reduced nonspecific effects. The company also makes genes, oligos with specialty bases, exceedingly long oligos, and diagnostic kits. All oligos are made at primer grade purity without extra charge. Also offered are level I and level II HPLC purification of oligonucleotides, in addition to research-grade HPLC purification. 800-888-2358 www.oligosetc.com PerSeptive Biosystems PerSeptive Biosystems offers both the PNA chemistry and a custom oligo synthesis service. PNAs are typically prepared on a 2 µmole scale, which yields ample product for majority of applications, and larger (25 µmole) scale preparations. PNAs may be routinely labeled with biotin, fluorescein, and rhodamine; most other specialty labels, e.g. cyanine and ABD dyes, may also be incorporated. A hydrophilic spacer is available for attachment of labels to PNAs prepared on resin. Mixed base sites are available for degenerate probe preparation. Amino acids may be incorporated at either end of the oligomers to aid in solubility, cellular uptake, or antibody binding. Each PNA is provided with analytical RP-HPLC and MALDI-TOF MS to insure synthesis fidelity. PNA may also be purified by RP-HPLC upon request. The custom oligo service can currently be ordered through PerSeptive Biosystems, but by June 30th will be available through the Applied Biosystems Custom Oligo Service Web Site. That site can be found at http://oligos.abd.perkin-elmer.com/oligo/worldwide.html. 800-899-5858 www.pbio.com Solid Phase Sciences Solid Phase Sciences specializes exclusively in the custom synthesis of fluorogenic oligonucleotides. The company offers both dual and single labeled probes with a variety of fluorogenic quenchers and reporter groups. The standard probes have a single isomer 6-fluorescein at the 5 ´ -end and single isomer TAMRA at the non-extendible 3 ´ -end. Solid Phase Sciences also supplies resins and reagents necessary for researchers to prepare their own modified oligonucleotides. The pricing for its oligos is $275 for 50 nmole scale, $350 for 200 nmole scale, and $425 for 1 µmole scale. 800-436-6631 www.solidphase.com Synthegen Synthegen was one of the first companies to specialize exclusively in modified oligonucleotides. It offers a full range of modifications: phosphorothioate oligonucleotides, which can also be fluorescently tagged; modified bases; amine and thiol functional linkers; chemical modifications; hapten modifications; and a broad spectrum of fluorescent labels, including fluorogenic dye quencher probes. Synthegen offers a choice of four reporter dyes and two quencher dyes for fluorogenic probes, which are available in two formats. In the standard format, the reporter is attached to the 5in addition to research-grade HPLC purification. 800-949-8903 www.synthegen.com Synthetic Genetics Synthetic Genetics has specialized n the production of a wide array of DNA dye labels, modifications and purifications since 1985. Oligonucleotides are synthesized under GMP conditions using the latest DNA chemistries to avoid depurination and breakdown of modifications during cleavage and deprotection. Oligos are available in 50 nmole, 0.2 µmole, 1.0 µmole, 10 µmole, and larger scales. Synthetic Genetics offers both HPLC and PAGE purification to remove incomplete oligos, depending on the type of oligo and purity and yield requirements. (HPLC generally gives higher yields but lower purity than PAGE purification.) Unique HPLC purification schemes are available for alkaline phosphatase and horseradish peroxidase conjugated oligos, as well as dual-label fluorogenic probes. All custom syntheses include a technical data sheet and gel picture. Synthetic Genetics offers fluorescent dyes including Molecular Probe dyes, the ABI dyes FAM, TAMRA, HEX, TET and JOE in dual label configuration, and 40 dyes that can be placed on the 5´-, 3´- end or internally (free HPLC purification). Also offered are phosphorothioate, mixed base and RNA oligonucleotides, alkaline phosphatase and horseradish peroxidase enzyme conjugates, and dual label fluorogenic 5 ´ -nuclease assay probes, in standard configuration or with some reporter dyes or quencher dyes in different 5 ´ -, 3 ´ - or internal positions. These probes are also available in two scales, 0.2 µmole scale for testing, and 1 µmole scale for larger experiments. 800-562-5544 www.syntheticgenetics.com TriLink BioTechnologies, Inc., is entering its second year as a provider of specialized oligonucleotides and nucleosides. In the area of RNA and RNA analog oligos, TriLink offers 2 ´ -OMe analogs, as well as oligos with all four 2 ´ -fluoro nucleosides, which form stronger duplexes with DNA than the 2 ´ -OMe but are less resistant to nuclease degradation. Also offered are chimeric oligos, the most common of which have a phosphodiester or phosphorothioate central region surrounded by nuclease-resistant (with 2´-modified nucleotides, for example) arms. Uniquely TriLink's is a new class of antisense agents, employing N3 ´* P5 ´ phosphoramidite (pnODNs) from LYNX, and methylphosphonate oligos, the only oligos with a non-ionic, nuclease-resistant backbone. TriLink offers linkers and spacers, single and multiple conjugates, and radioactively labeled oligos (35S and 3H). If the modification you desire is not among TriLink's offerings, the company provides custom phosphoramidites, as well as custom nucleotide triphosphates. 800-863-6801 www.trilinkbiotech.com The author can be reached at ldefrancesco@the-scientist.com

Special Needs: Custom Houses that Provide Specialized Oligos

Interested in reading more?

Become a Member of