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Oligo synthesis: Why IDT leads the oligo industry

When customers order oligonucleotides from Integrated DNA Technologies (IDT), they receive the highest quality oligos in the market. Through improvements to traditional synthesis chemistries and advances in our proprietary synthesis platform, we can synthesize longer oligos with better sequence fidelity and higher purity than our competitors. Our technologies also allow us to produce other synthesis products (see sidebar, Product focus) that can be used in a number of molecular biology applications. Here, we provide a comprehensive walkthrough of our oligo manufacturing process, and highlight the key factors that contribute to our industry-leading products.

Quality starts with proprietary synthesis platforms

The exceptional quality of IDT products starts with the equipment we use during synthesis. While many oligo manufacturers use the same general synthesis method described below (see section, The phosphoramidite approach), IDT is unique in that almost everything we use to produce an oligo is made in our own facilities. We formulate all of the key synthesis reagents in-house, and we design, manufacture, and calibrate our own DNA synthesizers and manufacturing software. This vertical integration enables us to internally monitor equipment performance, make quick alterations, and optimize each step of the oligo synthesis process. This is an important advantage, as it yields higher quality and shorter turnaround times than manufacturers who often rely on third-party suppliers.

IDT has engineered multiple synthesis platforms to accommodate the diversity of our customer’s needs. For common, unmodified oligos, we use highly automated, high-throughput synthesizers ideal for rapid production and quick turnaround times. For nonstandard oligos, we use synthesizers optimized for enhanced flexibility. These synthesizers facilitate the addition of complex modifications, such as fluorophores, quenchers, linkers, spacers, and modified bases (see sidebar, Additional reading, Oligonucleotide modifications).

Our optimized solid support yields a highly efficient synthesis process

Though our synthesis platforms vary in speed and flexibility, they all use solid-phase synthesis to produce an oligo. This means oligos will be tethered to a solid surface while they are being made. At IDT, we engineer our solid supports in-house, and we can easily optimize their properties to accommodate oligo synthesis at different scales. This is one reason IDT is an industry-leader in oligo quality and performance.

Prior to oligo synthesis, each solid support is fixed with a phosphoramidite monomer—a chemically-modified DNA nucleotide (see sidebar, Phosphoramidite monomers). The modified nucleotide is covalently attached to the solid support through its 3′ carbon. As oligonucleotide synthesis proceeds in the 3′→5′ direction, it becomes the 3′-most nucleotide of the oligo.

Note: Initial oligo subunits are not limited to nucleotides A, C, T, and G. IDT can also fix modifications to the solid support when they are needed at the 3′ end of an oligo (see sidebar, Additional reading, Oligonucleotide modifications).

Figure 1. A cytosine phosphoramidite. While the use of protecting groups can vary among oligo manufacturers, dimethoxytrityl (DMT), shown here in red, is widely used to protect the reactive 5' hydroxyl groups on the 4 nucleotides (A, C, T, G).

Phosphoramidite monomers

Phosphoramidite monomers, or phosphoramidites, are normal DNA nucleotides—adenine, cytosine, thymine, guanine; A, C, T, G—that are chemically modified with protection groups. These protection groups prevent reactive amine, hydroxyl, and phosphate groups of the nucleotides from undergoing unwanted side reactions, and force the formation of the desired product during synthesis. While the specific protection groups applied to each nucleotide vary among manufacturers, the use of a dimethoxytrityl (DMT) group for 5′ hydroxyl protection is ubiquitous in the industry (see Figure 1). As we walk through the synthesis process in the following sections, we will use the term “nucleotides” in place of phosphoramidite monomers, but keep in mind this important distinction. Also note that phosphoramidites can be RNA nucleotides, including Uracil (U).

A refined method for oligonucleotide synthesis

After a solid support containing the correct initial nucleotide (or modification) has been placed on the synthesis platform, IDT begins manufacturing the DNA or RNA oligo using a widely-known method called phosphoramidite addition. Although many manufacturers employ this method, IDT uses chemical reagents that are formulated in-house and optimized to our unique manufacturing process. Our employees are a collaborative network of departments, dedicated to ensuring the most efficient synthesis with precise chemistry that uses the best possible reagents. This commitment to quality has led to our consistent production of industry-leading oligos year after year.

The phosphoramidite approach: Deblocking, coupling, capping, and oxidation

The synthesis process adds nucleotides one by one, using a repeated 4-step cycle of deblocking, coupling, capping, and oxidation for each A, C, T, or G addition. Figure 2 outlines this process.

Figure 2. Overview of the phosphoramidite approach. This 4 step cycle repeats until the oligo receives its final nucleotide. The DMT protecting group (DMTO) is shown in red. IDT uses this method to manufacture all our oligos.


Step 1: Deblocking. 3′→5′ nucleotide addition requires initial removal of the dimethoxytrityl (DMT) protecting group on the 5′ carbon of the receiving oligo. This is accomplished by treating the oligo with a specialized deblocking reagent, which leaves the oligo with a free 5′ hydroxyl group that can then react with the next nucleotide. This step is also known as detritylation, or deprotection.

Step 2: Coupling. After deblocking, the support-bound oligo is ready to couple with the next nucleotide in the oligo sequence. As a new nucleotide is introduced to the oligo, it reacts with a weak acid to form a “phosphoramidite intermediate”. This conversion allows nucleotide interaction with the unblocked hydroxyl group on the 5′ end of the receiving oligo, resulting in its covalent attachment through a phosphite-triester bond.

Step 3: Capping. Despite many improvements to the phosphoramidite approach (see sidebar, A brief timeline of oligo synthesis), 100% coupling efficiency never possible. Even with precise chemistry and pure reagents, some support-bound oligos will not couple with the added nucleotide, leaving a free 5′ hydroxyl group. If not blocked, this reactive group can couple to the nucleotide added in the next cycle, producing an oligo with a deletion in its sequence. To avoid this, we perform a capping step that prevents these molecules from further extension.

IDT caps the free 5′ hydroxyl groups by adding a unique acetylating reagent that renders them inert. Unable to participate in any subsequent reactions, capped oligos remain at their incomplete length for the duration of the synthesis process. The collection of capped oligos in the final sample, referred to as “truncated product”, can be removed by additional purification (see section, Oligo purification: PAGE and HPLC). While the presence of some truncated molecules in the final oligo product is inevitable for any oligo manufacturing company, the goal is to minimize them. Our synthesis process consistently achieves extremely high coupling efficiencies (>99%), resulting in high quality oligos with low levels of truncated product (see section, IDT coupling efficiencies).

Step 4: Oxidation. Once unreacted oligos have been capped, the phosphite-triester linkage between a growing oligo and its newly-bound nucleotide must be stabilized. We accomplish this by adding a unique mixture of iodine and water into the synthesis platform. The mixtures oxidizes the phosphite into phosphate, resulting in a stable phosphor-triester bond between the oligo and its new nucleotide.

Repeated cycling, final processing, and desalting

Phosphoramidite addition repeats until the desired sequence is complete. Once the last nucleotide is attached, oligos undergo a process to remove the many protecting groups that are still attached. This includes a deblocking step to remove the DMT group attached to the final nucleotide, as well as other chemical reactions for the various nucleotide-specific protection groups (see sidebar, Phosphoramidite monomers). After these groups are removed, oligos are cleaved from the solid support, collected in a holding plate, and desalted to remove any additional contaminants. The final product is a functional single-stranded DNA molecule, ready for further purification (if needed), followed by IDT quality control.

Oligo purification: PAGE and HPLC

Longer oligos, modified oligos, and oligos used in particularly demanding applications, may require further purification to ensure that remaining truncated products do not compromise downstream reactions. IDT purifies such oligos using polyacrylamide gel electrophoresis (PAGE) and/or high performance liquid chromatography (HPLC).

PAGE. PAGE purification separates full-length product (FLP) from shorter species with great efficiency. It is most effective for unmodified oligos that only need truncated product removed. PAGE purification substantially reduces the amount (mass) of final oligo product, but most customers see the dramatic increase in purity as an acceptable trade.

RP and IE HPLC. HPLC purification, a form of column chromatography, can be done one of two ways. Reverse-phase (RP) HPLC separates oligos based on their affinity for particular solvents, while ion-exchange (IE) HPLC separates oligonucleotides based on their charge. IDT uses HPLC for modified oligos that include additions such as linkers, spacers, non-standard bases, and hydrophobic modifications. The type of HPLC used is dependent on the particular sample. Similar to PAGE, HPLC-purified oligos will undergo an unavoidable loss of mass, but this is offset by the gain in purity.

Regardless of length, IDT recommends that researchers consider additional purification (PAGE/HPLC) for any oligo used in demanding applications such as site-directed mutagenesis, cloning, and gel-shift protein-binding assays. Our support team is available to provide purification recommendations. Contact them at custcare@idtdna.com.

IDT quality control: ESI-MS, CE, and HPLC

Oligo purification does not provide information about oligo quality. We understand how important it is to produce a product that delivers consistent results to its customers, which is why we dedicate valuable time and resources to ensuring that the oligos we produce are the correct sequence and include a high proportion of full-length product.

ESI-MS. For sequence verification quality control, IDT uses electrospray ionization (ESI) mass spectrometry. The technique measures the final mass of an oligo, which is then compared to the expected mass based on the specific oligonucleotide sequence. Thus, if an oligo is missing a nucleotide, contains an extra nucleotide, or contains an incorrect nucleotide, it will be evident here. While many oligo manufacturers use matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry to measure oligonucleotide mass, our extensive experience has shown that this method becomes less accurate for oligos >40 nt and is ineffective for long oligos >75 nt. ESI-MS, however, can accurately measure oligos up to 200 nt. All synthesized oligos at IDT undergo ESI-MS for sequence verification, and we provide this quality control data to our customers with every order they place.

CE. Capillary electrophoresis (CE) is a quality control measure we use to determine the percentage of full-length product relative to truncated product. The method allows us to monitor the performance of our synthesizers and purification methods and is also used to provide customers with purity guarantees. IDT provides complementary purity guarantees and CE analysis for purified, unmodified oligos <60 nt that have low secondary structure. For all other oligos, customers can receive CE analysis for their order by purchasing a purity guarantee separately.

Analytical HPLC. Certain oligos possess secondary structures that can lead to anomalous CE results. For these oligos, we use analytical HPLC to assess their purity—this can be done with both RP and/or IE HPLC, depending on the oligo’s properties. Additionally, IDT will use analytical HPLC for quality control on other oligos if specifically requested by the researcher.

IDT coupling efficiencies show industry-leading consistency

Chemical and physical restraints reduce coupling efficiency to <100% during each cycle of synthesis (see section, Step 3: Capping). As a result, the small percentage of truncated product (<1%), generated with every nucleotide addition, accumulates. Thus, as sequence length increases, so does the total percent of truncated product. A 25mer oligo with 99.5% coupling efficiency will have an estimated 88.6% FLP (0.99524), while a 50mer with the same coupling efficiency will have an estimated 77.8% FLP (0.99549). The relationship between oligo length, coupling efficiency, and FLP percentage is shown in Figure 3.

Figure 3. The general relationship between FLP yield, oligo length, and various coupling efficiencies. Small decreases in coupling efficiency (≤1%) result in large decreases in FLP yield, most notably for long oligos.


IDT’s proprietary synthesis process not only achieves extremely high coupling efficiencies, but does so with a consistency that is not seen elsewhere in the industry. We understand the value in a reliable product, one that will perform the same each time it is used. Many customers use our oligos in lengthy projects that require multiple purchases over time. In these situations, consistency is absolutely crucial. Figure 4 illustrates our commitment to consistent coupling efficiencies.

Figure 4. IDT coupling efficiencies are high and consistent. Oligos from the IDT Coralville manufacturing facility were collected every other day for 4 months (July-Nov 2015) and evaluated for coupling efficiency. Coupling efficiency showed little variation, remaining at 99.25-99.35% for 20-30 nt oligos, and at 99.3%-99.4% for 30-50 nt oligos.


IDT: The global leader in nucleic acid synthesis

Due to our proprietary manufacturing processes and dedication to exceptional quality control, IDT has become the largest supplier of custom nucleic acids in the world, serving sectors of academic research, biotechnology, clinical diagnostics and pharmaceutical development. All based on the oligo synthesis technologies described here, IDT products support a wide variety of applications, including next generation sequencing (NGS), DNA amplification, genotyping and SNP detection, genome editing, expression profiling, gene quantification, synthetic biology, and functional genomics. Today, IDT synthesizes and ships an average of 44,000 distinct oligos per day to more than 82,000 scientists worldwide. IDT manufacturing locations include facilities in Coralville, Iowa; San Diego, California; Leuven, Belgium; and Singapore. For more information about our unique processes and products, visit us at www.idtdna.com.

A brief timeline of oligo synthesis

1953: Watson and Crick’s landmark paper reveals the double helix structure of DNA. (Watson, Crick 1953)

1956-1959: Dr Har Gobind Khorana and colleagues introduce the phosphodiester method of oligonucleotide synthesis—a protocol for coupling phosphorylated nucleotides using a 3-step cyclic scheme and dicyclohexylcarbodiimide (DCC). (Khorana 1956, 1959)

1961-1963: Members of the Khorana lab introduce 5′ hydroxyl protection with dimethoxytrityl (DMT), as well as the nucleotide-specific protecting groups that are still in use today. (Smith 1961, Schaller 1963)

1965: Dr Robert Letsinger publishes the first paper describing solid-phase synthesis of oligonucleotides. (Letsinger, Mahadevan 1965)

1969: Letsinger publishes the first paper on phosphotriester synthesis, an improvement from Khorana’s phosphodiester method, and one that could be replicated in many labs. (Letsinger, Ovilkie 1969)

1970: Khorana’s findings lead to the synthesis of an active 72 nt tRNA molecule. (Agarwal 1970)

1975-1976: Letsinger introduces phosphite-triester synthesis, including the final oxidation step and formation of a stable DNA backbone. (Letsinger 1975, 1976)

1983: The Caruthers lab introduces the phosphoramidite approach, based on tetrazole-activated phosphoramidite monomers that are made in advance and stored until synthesis. (McBride 1983)

1987: Dr Joseph Walder partners with Baxter Healthcare Corporation to found Integrated DNA Technologies.

References:

Integrated DNA Technologies. (2011) Chemical synthesis of oligonucleotides. [Online] Coralville, Integrated DNA Technologies. Available athttp://www.idtdna.com/Pages/docs/technical-reports/chemical-synthesis-of-oligonucleotides.pdf [Accessed 6 Oct, 2015].

Brown T and Brown T, Jr. (2005-2015) Solid-phase oligonucleotide synthesis. [Online] Southampton, UK, ATDBio. Available at http://www.atdbio.com/content/17/Solid-phase-oligonucleotide-synthesis [Accessed 6 Oct, 2015]

Hogrefe R. (2015) A Short History of Oligonucleotide Synthesis. [Online] San Diego, TriLink BioTechnologies. Available athttp://www.trilinkbiotech.com/tech/oligo_history.asp [Accessed 8 Oct, 2015].

Watson JD and Crick FH. Molecular structure of nucleic acids: A structure for deoxyribose nucleic acid. Nature. 1953 Apr 25; 171(4356):737–738

Khorana HG. Synthesis and structural analysis of polynucleotides. Jour Cell Phys. 1959 Dec; 54(1):5–15

Khorana HG, Tener GM, et al. A new approach to the synthesis of polynucleotides. Chem and Ind. 1956; 1523

Smith M, Rammler DH, et al. Synthesis of uridylyl-(3"→5")-uridine and uridylyl-(3"→5")-adenosine J Am Chem Soc. 1961; 84:430–440

Schaller H, Weimann G, et al. Protected derivatives of deoxyribonucleosides and new syntheses of deoxyribonucleoside-3″ phosphates. J Am Chem Soc. 1963; 85:3821–3827.

Letsinger RL and Mahadevan V. Oligonucleotide synthesis on a polymer support. J Am Chem Soc. 1965 Aug 5; 87:3526–3527

Letsinger RL, Ogilvie KK, et al. Synthesis of oligothymidylates via phosphotriester intermediates. J Am Chem Soc. 1969 June; 91(12):3350–3355, 3360–3365

Agarwal KL, Büchi H, et al. Total synthesis of the gene for an alanine transfer ribonucleic acid from yeast. Nature. 1970 July 4; 227:27–34

Letsinger RL, Finnan JL, et al. Phosphite coupling procedure for generating internucleotide links. J Am Chem Soc. 1975 May; 97(11):3278–3279

Letsinger RL and Lunsford WB. Synthesis of thymidine oligonucleotides by phosphite triester intermediates. J Am Chem Soc. 1976 June; 98(12):3655–3661

McBride LJ and Caruthers MH. An investigation of several deoxynucleoside phosphoramidites useful for synthesizing deoxyoligonucleotides. Tetrahedron Letters. 1983; 24(3):245–248

Product focus

Additional nucleic acid products generated from IDT oligos:

Additional reading

Oligonucleotide yield, resuspension, and storage

Your oligos have arrived! Do you know how to resuspend and store them? Here are some in depth guidelines and recommendations.

Oligonucleotide modifications: Choosing the right mod for your needs

Read these suggestions for selecting oligonucleotide modifications, and a discussion of how they can help you in your research.

Which biotin modification to use?

Learn about the applications for each of the different biotins available through IDT.

Author: Nolan Speicher is a scientific writing intern at IDT

© 2015 Integrated DNA Technologies. All rights reserved. Trademarks contained herein are the property of Integrated DNA Technologies, Inc. or their respective owners. For specific trademark and licensing information, see www.idtdna.com/trademarks.



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