Oligonucleotide API Manufacturing | Agilent
Oligonucleotide API Manufacturing ..
The Agilent in situ microarray synthesis ..
Oxidation and detritylation reactions were carried out by flood steps in a flowcell as depicted in . The Agilent flowcell is constructed such that a glass substrate carrying the microarrays forms one wall of the reactor chamber. The substrate is brought to bear upon a seal embedded in the perimeter of the fixed reactor cell thus forming a high aspect ratio sealed chamber, where the aspect ratio is defined as the ratio of the planar flowcell width L to the lateral gap height h. Typically, the planar width of the substrate is 15–20 cm while the gap is 0.5–1 mm. Active liquid reagents, wash solvents and gases are introduced into the flowcell through two ports. One port is located at the bottom corner of the cell and one at the top. A series of solenoid valves control the inflow and outflow of reagents to these two ports. The flowcell is mounted such that the walls of the flowcell are vertical so that gravity assists draining. During a typical synthesis cycle, the reagents are first introduced into the flowcell from the bottom port until the flowcell is filled (fill time). The reagents are then left in the flowcell without mixing for 30 s (oxidation) and 60 s (detritylation). We define these periods (when the flow within the flowcell is stationary) as the dwell time. Finally, the reagents are drained from the bottom port (drain time), followed by washes using two flowcell volumes of acetonitrile (ACN). This is typically 30–50 ml and is dependent upon the particular flowcell geometry. In the depurination controlled detritylation process, the detritylation solution is driven out of the flowcell through the top port of the flowcell using an inflow of oxidation solution through the bottom port. The plumbing connected to the outlet port of the flowcell is carefully constructed to avoid excessive restriction to the exiting liquid.
Interestingly we also observed that the comparative results of microarray hybridization analysis did not correlate with those of Solexa sequencing due to specific consensus sequences that sequenced poorly. The oligonucleotides not seen by sequencing were identified in substantial amounts by microarray hybridization. Together with the relatively low PCR error rate of the combined PCR “read-off” microarray and subsequent PCR amplification this demonstrates that the “read-off” approach is not sequence dependent but that the Solexa sequencing is. Similarly, significant skewing has previously been reported in Solexa sequencing of a PCR-amplified synthetic oligonucleotide library , perhaps suggesting that comparative mRNA profiling analysis on Solexa needs to be done with care.
Agilent expands oligonucleotide manufacturing services
A detailed study of fluid mechanics within the Agilent flowcell was undertaken in order to understand and control depurination within the microarray synthesis process. This article describes a new chemical strategy to optimize reagent flows so that oligonucleotide depurination is not only controlled but also minimized. This new depurination-controlled process is utilized to synthesize oligonucleotides of up to 150 nucleobases in length on microarrays. The success of the depurination-controlled process was confirmed by PCR, sequence analysis, and direct 5′-end labeling followed by PAGE.
When studying depurination within a flowcell reactor, one naturally refers to the vast databank of depurination on CPG (26–29). However, oligonucleotide synthesis within a CPG column differs greatly from oligonucleotide synthesis within a flowcell reactor. The most obvious difference is that the phosphoramidite coupling step has been removed to a specialized print chamber when synthesizing oligonucleotides on microarrays. The synthesis surface is also very different. While CPG is a 3D surface, that maintains a uniform surface characteristic throughout oligonucleotide chain extension, the glass substrates of the microarray process are typically flat, non-porous solid surfaces that generate a patterned array of oligonucleotides. The Agilent microarray flowcell also has a small surface area covered by a large reagent volume, typically holding 20 ml. This is the polar opposite of a 1 µmol CPG column, which has a large surface area covered by a small reagent volume (∼0.4 ml). The smaller capacity of the CPG column means that less reagent is used per chemical step and that multiple column volumes of washing solution can be used between chemistry steps. The high volume requirements result in the flowcell supplying, at best, one to two flowcell volumes of washing solution between chemistry steps.
an oligonucleotide library for synthesis .
We have achieved the ability to synthesize thousands of unique, long oligonucleotides (150mers) in fmol amounts using parallel synthesis of DNA on microarrays. The sequence accuracy of the oligonucleotides in such large-scale syntheses has been limited by the yields and side reactions of the DNA synthesis process used. While there has been significant demand for libraries of long oligos (150mer and more), the yields in conventional DNA synthesis and the associated side reactions have previously limited the availability of oligonucleotide pools to lengths
Of interest was that the 9976 sequences were seen between 1 to 4837 times each (). This significant difference in the number of reads of each oligonucleotide was initially thought to correspond to an unexpected large difference in the actual amount of the respective oligonucleotide in the library. Closer examination of the sequences revealed that the oligonucleotides that had poor frequencies of observation had the same consensus sequences as the non-identified oligonucleotides (). It is important to note that all of the oligonucleotides not seen by sequencing were observed by microarray hybridization in substantial amounts (the arbitrary microarray intensities where in the range of 6,000–38,000 compared to the full intensity range of 2,700–55,000; and ArrayExpress: E-MEXP-3102). Thus, no evidence of low synthesis rate of the high GC-content CGC-codon was observed in the microarray hybridization experiment and the low observation frequency of CGC containing oligonucleotides in Solexa sequencing cannot be explained by low synthesis efficiencies.
incorporate Agilent’s Oligonucleotide Library Synthesis ..
Partner with Agilent’s Nucleic Acid Solutions Division ..
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Nucleic Acid Analysis | Agilent
Another application of the technique could be the synthesis of defined siRNA libraries by employing an RNA polymerase rather than DNA polymerase, which would allow pools of siRNA to be synthesized from DNA microarrays –. Again, masking could allow rapid generation of separate oligonucleotide pools and the array to be re-used.
Content Type Certificate of Analysis (26049) ..
This technique offers efficient and inexpensive generation of thousands of defined oligonucleotides, which could allow the rapid synthesis of specific primers for use in genome sequencing and genotyping assays or DNA-encoding methods and aptamer screening. Furthermore, this method gives easy access to unpurified mixtures of microarray-synthesized oligonucleotides, which have been used directly in generation of high-quality gene assembly . This technique could also allow production of defined DNA libraries by employing an appropriate microarray design. For example, a microarray with 100 defined subarrays, each with repeats of a single oligonucleotide, would enable synthesis of separate oligonucleotide pools simply by using a cover-slip with 100 separate chambers .
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The Agilent in situ microarray synthesis process has been designed for the synthesis of high-quality nucleic acids and is built on two fundamental technologies. First, inkjet printing within an anhydrous print chamber allows for the spatial control of the phosphoramidite coupling step, which results in the synthesis of thousands of unique sequences on a 2D planar surface. Second, a flowcell reactor within the Agilent microarray synthesizer is utilized for the remaining chemistry steps. Oxidation and detritylation reactions are carried out in the flowcell by immersion, or flooding, of the growing oligonucleotides with the respective oxidation and detritylation reagents. While the standard Agilent microarray consists of oligonucleotides 60 nucleobases in length, printing oligonucleotides exceeding 100 nucleobases would appear to be attainable considering the high stepwise coupling step with Agilent’s print chamber. Such yields are the result of jetting small volumes of highly concentrated reactants onto an oligonucleotide chain growing from a solid surface. The excess concentration of the phosphoramidite and tetrazole solutions is further enhanced because the quantity of DNA within each spatially controlled feature does not exceed the femtomolar range. The added benefit of a proprietary anhydrous chamber ensures that moisture is kept to a minimum, allowing the coupling reaction to proceed more efficiently. With such reproducibly high coupling yields, the traditional capping step has been eliminated from the oligonucleotide synthesis cycle. Given the successful optimization of stepwise coupling yield with the Agilent platform, the next step toward the synthesis of oligonucleotides exceeding 100 nucleobases is minimization of depurination.
Microarray Generation of Thousand-Member Oligonucleotide ..
Unless otherwise indicated, DNA microarrays were manufactured according to the legacy Agilent manufacturing process as described elsewhere (12,18,30,31). Overall, an automated tool designed by Agilent Technologies was used to enable the standard phosphoramidite chemistry on a silylated 6.625 × 6 in. wafer using the following major modifications. First, the solid support was a flat, non-porous surface rather than a locally curved, porous surface. Second, the coupling step was controlled in space using inkjet-printing technologies to deliver the correct amount of activator and phosphoramidite monomer to the appropriate spatial location on the solid support. Third, the oxidation and detritylation reactions were performed in dedicated flowcells whose mechanical operations are described in the next paragraph. Oxidation solution was with 0.02 M I2 in THF/pyridine/H2O and detritylation solution used 3% dichloroacetic acid (DCA) in toluene. Deprotection and cleavage of the DNA from the surface was performed as described by Cleary et al. (). Oligonucleotides were recovered after cleavage by lyophylization in Eppendorf tubes. Each array could contain up to 22 575 features. When fewer oligonucleotides were desired, the appropriate number of locations were left blank.
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