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custom oligo synthesis, oligonucleotides ..

This unit augments the detailed instructions provided by the manufacturersof oligonucleotide synthesizers.

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Oligonucleotide synthesis - dnareplicationsystem - …

Instruments take varying amounts oftime to synthesize oligonucleotides depending on the number of columnsin use, the length of the oligonucleotide, and the synthesis program.

A well organizedoverview of synthetic oligonucleotide synthesis, purification and quantitation

Oligonucleotide synthesis is carried out by a stepwise additionof nucleotide residues to the 5'-terminus of the growing chainuntil the desired sequence is assembled. Each addition is referredto as a synthetic cycle (Scheme 6) and consists of four chemicalreactions:

TriLink | A Short History of Oligonucleotide Synthesis

The obvious advantageof such a method is that doped and clean sequences can be synthesized onthe same oligonucleotide.

One may visualize an oligonucleotide microarray as a miniaturemulti-well plate where physical dividers between the wells (plasticwalls) are intentionally removed. With respect to the chemistry,synthesis of oligonucleotide microarrays is different from theconventional oligonucleotide synthesis in two respects:

Oligonucleotides are chemically synthesized using nucleosidephosphoramidites. A nucleoside phosphoramidite is a derivative ofnatural or synthetic nucleosides with protection groups added toits reactive exocyclic amine and hydroxy groups. As mentionedearlier (see Phosphodiester synthesis above), the naturallyoccurring nucleotides (nucleoside-3'- or 5'-phosphates) areinsufficiently reactive to afford the synthetic preparation ofoligonucleotides. A dramatically more reactive N,N-diisopropylphosphoramidite group is therefore attached to the 3'-hydroxy groupof a nucleoside to form nucleoside phosphoramidite. To preventundesired side reactions, all other functional groups ofnucleosides have to be rendered unreactive (protected) by attaching. Upon the completion ofthe oligonucleotide chain assembly, all the protecting groups areremoved. Below, the protecting groups currently used incommercially available and most common nucleoside phosphoramiditebuilding blocks arebriefly reviewed:

Custom Oligonucleotide Synthesis

In the 1970s, substantially more reactive P(III) derivatives ofnucleosides, 3'-O-chlorophosphites, were successfully usedfor the formation of internucleosidic linkages. Thisled to the discovery of the triester methodology. The group ledby M. Caruthers took the advantage of less aggressive and moreselective 1H-terazolidophosphites and implemented themethod on solid phase. Veryshortly after, the workers from the same group further improved themethod by using more stable nucleoside phosphoramidites as buildingblocks. Theuse of 2-cyanoethyl phosphite-protecting group in place of a lessuser-friendly group led to the nucleosidephosphoramidites currently used in oligonucleotide synthesis (seePhosphoramidite building blocks below).Many later improvements to the manufacturing of building blocks,instrumentation, and synthetic protocols made the phosphoramiditechemistry a very reliable and expedite method of choice for thepreparation of synthetic oligonucleotides.

In contrast to organic solid-phase synthesis and , the synthesis of oligonucleotides proceeds best onnon-swellable solid supports. The two most often used solid-phasematerials are Controlled Pore (CPG) and macroporous (MPPS).

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  • Peptide Conjugates of Oligonucleotides: Synthesis ..

    The term “oligonucleotide” or “oligo” usually refers to a synthetic laboratory-made DNA or RNA strand.

  • Solid-phase oligonucleotide synthesis - ATDBio

    Oligonucleotides are made by the sequential synthesis of nucleic acid biomolecules

  • Sigma-Aldrich Online Catalog Product List: Cap A

    The quality of your analysis starts with high fidelity oligonucleotide synthesis

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Bio-Synthesis, Inc. - Contact Us

In the 1950s, and co-workers developed a method where3’-O-acetylnucleoside-5’-O-phosphate2 was activated withN,N’-dicyclohehylcarbodiimide (DCC) or4-toluenesulfonylchloride (Ts-Cl) and a 5’-O-protectednucleoside 1 was reacted with the activatedspecies to give a protected dinucleoside monophosphate3. Uponthe removal of 3’-O-acetyl group using base-catalyzedhydrolysis, further chain elongation was carried out. Followingthis methodology, sets of tri- and tetradeoxyribonucleotides weresynthesized and enzymatically converted to longer oligonucleotides,which allowed elucidation of the . The major limitation of thephosphodiester method consisted in the formation of pyrophosphateoligomers and oligonucleotides branched at the internucleosidicphosphate. The method seems to be a step back from the moreselective chemistry described earlier; however, at that time, mostphosphate-protecting groups available now had not yet beenintroduced. The lack of the convenient protection strategynecessitated taking a retreat to a slower and less selectivechemistry to achieve the ultimate goal of the study.

TriLink BioTechnologies - Oligo Synthesis, mRNA, …

To make the solid support material suitable for oligonucleotidesynthesis, non-nucleosidic linkers or nucleoside succinates arecovalently attached to the reactive amino groups in AminopropylCPG, LCAA CPG, or Aminomethyl MPPS. The remaining unreacted aminogroups are capped with . Typically, threeconceptually different groups of solid supports are used.

Emerging Oligonucleotide Therapeutics - Discovery On …

In the early 1950’s, ’s group pioneeredH-phosphonate and triester methods of oligonucleotidesynthesis. Thereaction of compounds 1 and 2 toform H-phosphonate diester 3 is an H-phosphonatecoupling in solution while that of compounds 4 and5 to give 6 is a phosphotriestercoupling (see Phosphotriester synthesis below).

Oligo Entry - Integrated DNA Technologies

Oligonucleotide phosphorothioates (OPS) are modifiedoligonucleotides where one of the oxygen atoms in the phosphatemoiety is replaced by sulfur. Only the phosphorothioates havingsulfur at a non-bridging position as shown in Figure are widelyused and are available commercially. The replacement of thenon-bridging oxygen with sulfur creates a new center of at . In a simplecase of a dinucleotide, this results in the formation of a pair of Sp- andRp-dinucleoside monophosphorothioates whose structuresare shown in Figure. In a n-mer oligonucleotide where all(n - 1) internucleosidic linkages are phosphorothioatelinkages, the number of diastereomers m is calculated asm = 2(n - 1). Being non-naturalanalogs of nucleic acids, OPS are substantially more stable towards by , the class of that destoy nucleicacids by breaking the bridging P-O bond of the phosphodiestermoiety. This property determines the use of OPS as antisenseoligonucleotides in and applications where the extensive exposure tonucleases is inevitable. Similarly, to improve the stability of , at leastone phosphorothioate linkage is often introduced at the 3'-terminusof both and antisensestrands. In chirally pure OPS, all-Sp diastereomers are more stableto enzymatic degradation than their all-Rp analogs.However, the preparation of chirally pure OPS remains a syntheticchallenge. In laboratory practice, mixtures of diastereomers of OPSare commonly used. Synthesis of OPS is very similar to that ofnatural oligonucleotides. The difference is that the oxidation stepis replaced by sulfur transfer (sulfurization) and that the cappingstep is performed after the sulfurization. Of many reportedreagents capable of the efficient sulfur transfer, only three arecommercially available:

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