Editorial Article
Monday June 14, 2010
by Caitlin Smith
Who would have thought that the humble nucleic acids described decades ago by Watson and Crick would one day be eyed as therapeutic or diagnostic agents world-wide? With customized modifications, today’s oligonucleotides are doing more than probing blots in the lab. And in many cases, buying is cheaper than making your own.
“The most exciting new development in our field is the rapid movement towards genetic testing, which includes molecular diagnostics, companion diagnostics, and personalized medicine,” says Grace Lean, marketing associate at Biosearch Technologies. “In the past, oligos were manufactured at very small scales because academic researchers predominantly found use for them as tools in their research; however, oligos have become more significant in today’s society as they gain more traction in environmental and public health. Oligos are now in higher demand at larger scales and for repeated use.” Indeed, Lean says that biotech and pharmaceutical companies are commercializing oligos for human diagnostics, such as drug susceptibility and resistance. For example, Biosearch’s own “BHQ® (Black Hole Quencher®) Probes are currently used to test HIV drug resistance in South Africa,” says Lean. As oligos enter the human diagnostic market, a field highly regulated by government agencies, manufacturers must produce them under stringent conditions to ensure a consistently made product. Biosearch offers GMP manufacturing services that specialize in the production of oligo components for in vitro diagnostics including Analyte Specific Reagents (ASR) and other classes of diagnostic tests.
A systems approach for molecules
With considerable experience in custom nucleic acid production, Sigma-Aldrich is exploring systems approaches to handling large amounts of nucleic acid data. “Current trends are for improved qPCR instrumentation, allowing for more precise measurements and collection of larger data sets,” says Tania Nolan, global manager of applications and technical support at Sigma-Aldrich. “The types of detailed analyses that come within reach put emphasis on careful handling and require systems approaches.”
Sigma-Aldrich is using more bioinformatics in oligo design to address new challenges. “Some of these are defined by the scale of the requirements—for example, primer sequences for an entire transcriptome, or provision of a unique barcode so that libraries generated for next-generation sequencing can be characterized,” says Nolan. “In order to address such questions we need to turn to our expert bioinformatitians. Other challenges are due to increasing demand for highly sensitive and specific assays for PCR and qPCR. In these cases, we may be required to identify homologous regions from over a thousand viral serotypes and then design a single assay to detect all serotypes equally and with high sensitivity. After working with our bioinformatics team to identify suitable regions, we then use a combination of oligo modifications to provide the best solution.”
Custom, modified oligos
The most exciting development in oligonucleotide synthesis today, according to J. Lynn Myers, VP of the Midland Certified Reagent Company, “is the ever increasing number of modifications that are being offered either by commercial suppliers of phosphoramidites or from individual labs that have synthesized a modified phosphoramidite for their own experiments.” Midland can incorporate more than one modification within a single sequence, and they offer endotoxin-free products too. “We offer sequences that contain locked nucleic acid (LNA) derivatives, whether it is just a single site within a sequence or the entire sequence is modified,” says Myers. “We have the ability to synthesize multiple sequences containing LNA modifications simultaneously. We can also synthesize chimeras containing both LNA modifications and RNA, [or] LNA modifications as well as a great number of other modifications.”
Gene Link offers modifications to oligos too, their newest being adenylation. “Our latest product offering is custom 5’-adenylation (5’-App) of both DNA and RNA oligos for use in next-generation sequencing applications,” says Arthur Johnson, manager of operations and business development at Gene Link. He believes that for oligos, you get what you pay for. “Unfortunately, there is always some trade-off between the price of the oligo and its performance in a PCR reaction. Generally speaking, even PCR primers should undergo some kind of purification, beyond simple desalting, before use. The take-home message here is to use clean PCR primers. You’ll be happy you did.”
Biosearch Technologies offers a wide collection of custom, modified oligos to support various genetic methods such as oligo conjugates for bead-based assays, RPA probes for isothermal amplification, Methylene Blue-labeled oligos as electrochemical DNA sensors, dual-labeled probes for real-time qPCR, and BHQplus™ Probes for probe-based SNP genotyping. They also offer ValuPanel™ Reagents, which are defined panels of probes and primers used to discriminate H1N1 and seasonal flu viral strains. These panels are also powered by Biosearch’s BHQ Probes.
For protein studies, scientists can take advantage of oligos in comprehensive mutagenesis strategies. Blue Heron Bio offers variant libraries made up of individual, sequence-verified clones, each differing from the parent sequence at one or a few codons. “In a single experiment, researchers can test every possible amino acid change in an enzyme or every possible change in the variable regions of an antibody, each present in a single clone,” says Dorene Nielsen, director of sales/marketing at Blue Heron Bio. “These libraries provide the best possible starting point for directed evolution, since every assay provides an unequivocal data point on a specific change and all the changes are tested in the same protein context. This approach is particularly well-suited for projects where assay costs are high and speed is critical, such as antibody affinity maturation.”
Quality, longer oligos made easier
Applications that take advantage of oligos longer than 100 bases are receiving more attention lately. “One example is the solution hybrid selection method for enrichment of next-generation sequencing targets,” says Johnson. “Here, the length of ultra-long oligos provides specific advantages as capture probes, such as little or no allelic bias in the pool of enriched targets, and the ability to capture genomic targets containing large deletions.” Another new area is the use of ultra-long oligos as templates for generating three-dimensional DNA nanoassemblies. “Successful construction of such structures is critically dependent on the flexibility of the component parts,” says Johnson. “Ultra-long oligonucleotides can provide such flexibility, thereby expanding the size and type of 3-D nanostructures possible.”
Integrated DNA Technologies (IDT) offers a flexible way to customize oligos, including oligos up to 200 bases, in their Ultramers line, for applications such as site-directed mutagenesis, gene construction, next-generation sequencing, and ddRNAi. Ultramers can simplify applications, according to IDT qPCR and OEM Business Unit Leader Stephen Gunstream. “Using a sequence with a large stretch of tandem repeats for cloning is very difficult with traditional methods, but since Ultramers are directly synthesized, the sequence can be ordered and inserted into a clone very easily,” says Gunstream. “There are a number of customers that order Ultramers for site-directed mutagenesis. Traditional methods for mutagenesis require primer design, multiple reagent purchases and significant time in the lab.” Ultramers also work well as controls for qPCR and next-generation sequencing. “Instead of amplifying a template for a spike-in control, a customer can order the Ultramer directly and add it to the reaction,” explains Gunstream.
Blue Heron Bio has developed a new technology for assembling BAC-sized DNA fragments in E. coli, enabling them to make fragments of up to 250,000 base pairs. “This new technology allows our customers to design and source almost any mammalian gene or gene complex, the very longest cDNAs, and large segments of bacterial, plant, or yeast chromosomes,” says Nielsen. According to Nielsen, the biggest challenge in oligo design is that “most scientists have not yet begun to utilize the freedom that gene synthesis provides. With the availability of gene synthesis, scientists now need to start thinking, ‘How would I answer this question if I could use any DNA molecule, not just the one that happens to be in my freezer or available in the lab?’ Science will move faster and cost less when more scientists make this leap.”