Talk:Oligonucleotide synthesis

similarity

Phoshoramidite entry is similar to this but made indipendently, I think. It should be fixed as it contains some extra stuff but lacks other stuff and does not link here. —Preceding unsigned comment added by Squidonius (talk • contribs) 16:55, 25 October 2007 (UTC) This is what was cut out of phoshoramidite:

Calculation of Expected Yield== While regular phosphoramidite bases and special modifications will couple with varying yields, using the accepted efficiency of 98% per coupling reaction, one can estimate the yield for a given length of DNA to be synthesized. The expected yield (Y) is found by raising the average efficiency (98%) to the power of the number of couplings (N) to be performed.

Y=0.98^(N)

Solid phase chemical synthesis

Main article: Oligonucleotide synthesis

Solid phase chemical synthesis is done by protection chemistry where the most reactive groups are protected to avoid unwanted products. Whereas in biological processes nearly all enzymes work on DNA from the 5' to the 3' end, this artificial chemical process is done opposite of that. The 3' primer is immobilized via a linker onto a solid support (polystyrene beads or similar), allowing the chemicals to be added and washed off, while the 5' OH group is protected by a DMT (dimethoxytrityl) group. The free phosphoamidite bases have on their phosphate group a diisopropylamino (iPr₂N) group and a 2-cyanoethyl (OCH2CH2CN) group. The bases also have protecting groups on the exocyclic amine group (benzoyl or isobutyryl).

• Detritylation: The DMT is removed with an acid, such as TCA, resulting in a free OH
• Coupling: a phosphoramidite nucleotide is added (or a mix) and tetrazole which removes the iPr₂N group on the incoming base that is attacked by the deprotected 5'OH of the growing oligo (these reactions are not done in water but in tetrahydrofuran or in DMSO). In RNA the 2' is protected with a TBS (butyldimethylsilyl) group or with a Me group.
• Capping: The few (1%) free 5'OH must be stopped; they are capped with acetic anhydride and 1-methylimidazole.
• Oxidation: The phosphate group is made pentavalent by adding iodine and water. This step can be substituted with a sulphorylation step for thiophosphate nucleotides.

These 4 steps are repeated n number of times. The products are cleaved and deprotected (base and phosphate) thanks to base hydrolysis (ammonium hydroxide). They are then desalted and lyophilized or purified by HPLC. Reporter groups and so on are often added post-synthesis (aminoallyl groups are a common method).

Enzymatic synthesis

T4 RNA ligase and T4 DNA ligase are used for making longer oligos by ligating 2 short oligos (5'Phosphate donor and 3'OH acceptor). Template driven enzymatic synthesis is also efficient when using T7 Polymerase or the Klenow fragment and modified bases.

Microarrays

Main article: DNA microarray § Fabrication

An interesting development of this technology has allowed genechips to be made, where the probes are synthesised on the silicon chip, and not printed, allowing a higher resolution. This can be done via a mechanical mask where thin silicon rubber capillaries are put on a glass slide and the probes synthesised. More high-tech versions employ photolayable products and Photolithographic mask or micromirrors. The 1cm² surface of silicon is coated with a linker and a photoprotecting group such as nitroveratryloxycarbonyl is used and the mask exposes to a lamp the spots that will receive the subsequent nucleotide: this step is repeated for all four bases, but only one correct one is added to the growing probes on each spot (www.affymetrix.com). Thanks to digital light processing (DLP) technology (that give HD TVs) micromirrors were developed which have more detail and speed compared to masks, allowing the generation of microarray chips having one million and more features (www.nimblegen.com). DLP technology and improved synthesis chemistry is the basis for an extremely fast and flexible DNA microarray synthesizer, recently commercialized for cutting-edge research projects (www.febit.com).[1]

Uses==

• siRNA
• primers for PCR and RT PCR
• 70mers for printer microarrays
• 18mers in situ synthesis of probes for a genechip

drawning

There is a mistake in the drawing; an extraneous oxygen is present between the cytosine N1 and the ribose ring. —Preceding unsigned comment added by 140.247.246.12 (talk) 19:06, 9 April 2008 (UTC) FIXED thanks for notification that was a big one! --Squidonius (talk) 15:35, 13 April 2008 (UTC)

Good Article

I've listed this article as a good article as it meets the good article criteria. It is well-written, and has many reliable sources to confirm the statements presented. It is broad in its coverage, and addresses main aspects of the topic. Finally, it has diagrams to show the process. One more suggestion I'd make is to use numbered lists rather than bulleted lists for some parts. Best, -download | sign! 04:54, 26 April 2009 (UTC)

could we please have a short opening para for the nonexperts? this page made my brain hurt, i have passed at least one term of university chem and this lead section still made my eyes raw. "The lead ... should be written in a clear, accessible style ... to invite a reading of the full article." & please see WP:MOSINTRO "It is even more important here than for the rest of the article that the text be accessible." "In general, specialized terminology ... should be avoided in an introduction." "The subject should be placed in a context with which many readers could be expected to be familiar. ... Readers should not be dropped into the middle of the subject from the first word; they should be eased into it."

can i suggest something like

Oligonucleotide synthesis is the joining together of fragments of DNA and RNA in specific sequences. The process has been fully automated since the late 1970s. Upon the completion of the chain assembly, the product is released from the [Solid-phase synthesis|solid phase] to solution, [Protecting group|deprotected], and collected. The occurrence of side reactions sets practical limits for the length of synthetic oligonucleotides (up to about 200 nucleotide residues) because the number of errors accumulates with the length of the oligonucleotide being synthesized.[1] Typically, synthetic oligonucleotides are single-stranded DNA or RNA molecules around 15–25 bases in length. They are most commonly used as antisense oligonucleotides, small interfering RNA, primers for DNA sequencing and amplification, probes for detecting complementary DNA or RNA via molecular hybridization, tools for the targeted introduction of mutations and restriction sites, and for the synthesis of artificial genes.

and then—if anything i've cut from the original is not otherwise covered in the article below—put than in somewhere below the lead section. David Woodward ☮ ♡♢☞☽ 05:58, 6 September 2011 (UTC)

Sequence length

Has there been any progress in the length of sequence that can be synthesized? The article says:

The occurrence of side reactions sets practical limits for the length of synthetic oligonucleotides (up to about 200 nucleotide residues) because the number of errors accumulates with the length of the oligonucleotide being synthesized.

However, the reference is from 1992. Any progress since then? pgr94 (talk) 10:34, 25 June 2011 (UTC)

These days, the maximum length of synthetic oligonucleotides runs to about 300 nucleotide residues. There is no commercially available solid support material that allows the preparation of longer sequences. Also, it does not look like there is a strong driving force to increase the length. The majority of vendors and consumers seem to be more concerned with the throughput and the cost. I hope, this answers your question. Chemist234 (talk)

Thanks, it does indeed. pgr94 (talk) 13:22, 2 October 2011 (UTC)

Solid Support Material

Hello. In this article I read: "The two most often used solid-phase materials are Controlled Pore Glass (CPG) and macroporous polystyrene (MPPS).[55]" I checked the resource [55] and I havn't found anything about polysterene, it's just all about CPG. I need more informations about macroporous polystyrene (non-swellable polystyrene) for solid-phase oligonucleotide synthesis. I haven't even found it commercially available. I would appreciate some more information - even where to buy it or a CAS No - Thanks. — Preceding unsigned comment added by 134.100.29.35 (talk) 09:33, 18 September 2011 (UTC)

Hello. The macroporous polystyrene loaded with a variety of nucleosides and other linkers is commercialized by a number of companies. I understand that you are a small scale user. The most convenient option for you to purchase the loaded polystyrene is Glen Research (www.glenresearch.com). If you need unloaded macroporous aminomethyl polystyrene, you can contact AM Chemicals LLC (www.amchemicals.com). Best, Chemist234 (talk) 06:39, 2 October 2011 (UTC)

Thank you for your answer. I have used CPG with a loading of 30 µmol/g but I'm searching for a higher loaded solid-phase to make oligonucleotides on (not automated, manually). It would be nice to synthesize a small oligonucleotide on a solid-support with a loading of e.g. 200-1000 µmol/g. I know this is a high value and I don't know if it's possible to have such solid-phases. However, I think the highest possible loading of polystyrene is about 7 mmol/g but for the oligonucleotide synthesis you have to use non-swellable polystyrene so you can't achieve 7mmol/g. With a higher loading I would be able to see the compound in the nmr spectra, because I need the product in a milligram scale. Are there any solid-phases for oligonucleotide synthesis with a high loading? --92.224.124.118 (talk) 11:45, 27 November 2011 (UTC)

This should not be a problem. If you intend to record NMR of an oligo in solution, you can purchase high-loaded CPG500 (about 70-80 umol/g) or polystyrene loaded in a range of 200-400 umol/g. A 10 umol manual synthesis on either of the supports will yield about 15 mg of a purified 10-mer, which should be sufficient for your NMR experiments. If you intend to record NMR from a solid support-bound oligo, then you are limited to 31P spectra from a polystyrene-bound oligo. That works well, too. The loading of 200 umol/g will be sufficient to accumulate very decent FID's in 5 min. I hope this helps. Best, Chemist234 Chemist234 (talk) 01:27, 29 November 2011 (UTC)

Thank you for your answer. I'm interested in "polystyrene loaded in a range of 200-400 umol/g" for oligonucleotide synthesis. Can you tell me where to buy this? I've only found swellable polystyrene (with a loading: of 1 mmol/g). Edit: I found polystyrene 600-900 umol/g with a 1% divinylbenzene crosslinking (but this is a swellable support!). I guess I need a DVB crosslinkage of 20% (to make the support non-swellable). --134.100.204.166 (talk) 15:39, 29 November 2011 (UTC)

No PS with less than 60% crosslinking works in oligo synthesis. NittoPhase PS (see http://kinovate.com/products/nittophasehl), although quite expensive, is loaded up to 400 umol/g. Universal PS loaded at about 150 umol/g is available from http://amchemicals.com/Universal_SS.html. However, for your project, you do not have to use PS at all. Loaded CPG is substantially more friendly in handling, and the removal of DMT proceeds a lot faster. When the synthesis is manual, these properties make your life easier. To produce 20-50 mg of an oligo, it is sufficient to use CPG500 loaded around 70-80 umol/g. You can purchase it at http://www.glenresearch.com/ProductFiles/25-5040.html or at http://amchemicals.com/Universal_SS.html. Best, Chemist234 (talk) 03:59, 30 November 2011 (UTC)

Coupling Step: Influence of water

In Nucleoside_phosphoramidite I read that the phosphoramidite reacts with water to a H-phosphonate diester (see picture: [here]).

"When water is served as a nucleophile, the product is an H-phosphonate diester as shown in Scheme above. Due to the presence of residual water in solvents and reagents, the formation of the latter compound is the most common complication in the preparative use of phosphoramidites, particularly in oligonucleotide synthesis."

I have synthesized a modified phoshoramidite (as a crude product!) and I want to use this crude product for the coupling step without any further purification. In case that I have some H-phosphonate byproducts, will they also be coupled to CPG? Are H-phosphonates even soluble in acetonitrile or dichloromethane? What about hydrolized Bis-N,N’-diisopropylamino-(2-cyanoethyl)-phosphoramidite: will this compound also couple to CPG? --80.171.177.86 (talk) 14:49, 3 December 2011 (UTC)

None of the by-products will couple to the solid phase. If your phosphoramidite is soluble in MeCN, chances are that the respective H-phosphonate diester is soluble, too. With that said, I strongly recommend purifying your modified phosphoramidite by column chromatography on silica gel prior to the use in oligo synthesis. First, the coupling efficiency is always substantially higher with pure amidites than with the crudes, which means you will have to spend a lot less of your precious compound to obtain the same result. Second, in the (unfortunate) case that the coupling of a modified amidite does not work at all, you wouldn't know the reason for the failure because the use of a crude creates too many additional variables to consider. With the purified amidite, the choice is more limited and you can locate the failing step faster. In order to purify your amidite, suspend silica gel in 5% TEA in DCM (min 3 column vol), wait until the initially slightly warm suspension cools to RT, and pack the column. Equilibrate the column with 3% TEA in hexanes. It is likely that your amidite dissolves in toluene + TEA so that you can apply your mixture on the column as a solution. Next, elute the column with a step gradient of Et acetate in hexanes maintaining the constant conc. of 3% TEA. Nucleoside amidites elute at 60-97% Et acetate depending on the base. Most non-nucleosidic amidites are more mobile. After you collect fractions and evaporate the solvent, co-evaporate the amidite 3 times with anhydrous MeCN and dry on an oil pump. Finally, record ³¹P NMR spectrum in MeCN (not in CDCl3!!!) expecting to observe the peak (2 peaks if P is attached to a chiral moiety) between 145 and 150 ppm. A useful amidite is >90% pure by ³¹P NMR, but, most of the time, one obtains more pure material. The procedure described above works well as long as the amidite does not contain very base-labile protecting groups like Fmoc on a weak amino group or acyl protection on the phenolic OH. In those cases the losses are expected but can be minimized by reducing the concentration of TEA to 0.5-2%. For reproducible TLC, I recommend first eluting an empty plate with EtAc + 5% TEA, drying the plate by a heating gun, and then applying the spots of your amidite. Good Luck, Chemist234 (talk) 06:59, 7 December 2011 (UTC)

Thank you very much. Why do you first suspend silica gel in DCM and afterwards equilibrate the column with hexane? Why using DCM at all? You said nucleoside amidites elute at 60-97% Et acetate depending on the base. Do you have a reference (literature) for this? I would try it with thymidine. I already recorded some ³¹P NMR spectra in "dry" CDCl3, I think nucleoside amidites are stable enough in dry CDCl3 for several hours at least. However, I always have a peak around 14 ppm which must come from Bis-N,N’-diisopropylamino-(2-cyanoethyl)-phosphoramidite as well as two peaks at 0-10 ppm (maybe H-phosphonates). The peak at 14 ppm is big, I think its some sort of decomposed Bis-N,N’-diisopropylamino-(2-cyanoethyl)-phosphoramidite. I use 1.5 eq of it, and in the resulting ³¹P NMR spectrum (CDCl3) of the crude nucleoside amidite the integral is 0.5 (1.0 for the nucleoside amidite).--92.224.126.239 (talk) 19:55, 12 December 2011 (UTC)

Could you move your discussion to your talk pages? Thanks. Carstensen (talk) 16:30, 13 December 2011 (UTC)

Basicity of exocyclic amino group

Why is the basicity of the exocyclic amino group of guanosine low that it doesnt need to be protected (Source?)? Compared to Adenine and Cytosine the pK_A of Guanine is the highest with 12.3... Thet619 (talk) 23:29, 13 January 2013 (UTC)

• Since we are discussing oligonucleotides, we should consider pK_a of nucleosides or nucleotides rather than nucleic bases. For dG, there are several possible ionic equilibria. Thus, pK_a of 12.3 refers to dissociation of hydroxy groups in 2'-deoxyribose. Further, pK_a of 9.33 refers to ionization of O6 [equilibrium between N=C(OH)- and N=C(O^-)-] and illustrates the fact that the nucleic base in dG is a very weak acid. Protonation/deprotonation of amino groups is yet another equilibrium. The pK_a of N-protonated deoxyribonucleosides dG, dA, and dC is 2.4, 3.8, and 4.3, respectively. The pK_a of N-protonated ribonucleosides G, A, and C is 2.1, 3.6, and 4.1, respectively. In ribonucleoside-5'-phosphates, pK_a of pG, pA, and pC is 2.4, 3.74, and 4.5. This shows that, among all nucleosides, dG and G have the amino groups with the weakest basic properties. Chemist234 (talk) 10:40, 20 January 2013 (UTC)

Thx, now its clear. At least almost everything... In the article u were referring to the exocyclic groups, but in the answer u were talking about the C=N-C-groups (N7 of G, N1 of A and N3 of C) ... Do u have the pK_a-values of the exocyclic amino groups of nucleosides/nucleotides? I only found the values of the exocyclic groups of nucleic bases (pK_{a Primary} = 12.3 Guanine) Thet619 (talk) 22:11, 29 January 2013 (UTC)

• One cannot direct protonation specifically to the exocyclic amino group because it is not energeticaly favorable. The site of protonation is N7 for G, N1 for A, and N3 for C, as you correctly stated (see Blackburn, G. M. and Gait, M. J. Nucleic Acids in Chemistry and Biology. IRL Press, Oxford, New York, Tokyo, 1990, pp. 21-22 and Hall, R. H. The Modified Nucleosides in Nucleic Acids. Columbia University Press, New York, 1971). However, because the lone pair of exocyclic amino groups is delocalized into the aromatic system of the heterocyclic base, the net result is that, upon protonation, the nucleophilicity of the entire system including the exocyclic amino group becomes lower. Effectively, under these conditions the amino groups behaves as if it was protonated. The value of pK_a for guanine is irrelevant here. I hope it helps, Chemist234 (talk) 19:59, 30 January 2013 (UTC)

Too Technical?

@Download: I noticed that you deemed this a Good Article, but it seems far too technical to me. Do you have an opinion on this? Exercisephys (talk) 18:11, 14 March 2014 (UTC)

Hey Exercisephys, it's been several years since I evaluated this article, and unfortunately I must admit that I am not familiar at all with Good Article guidelines anymore. I'd value any additional input! -download ׀ talk 03:44, 15 March 2014 (UTC)

• @Exercisephys: Although this article might seem too technical, the nature of the subject in hand is such that either the article is technical, or it is useless. Many, if not most articles in natural sciences and mathematics are that way; their understanding requires more than a solid common sense plus a high school degree. See, for instance, any of the following Good Articles: Ascending cholangitis, Derivative, Dirac delta function, Johnson–Corey–Chaykovsky reaction, Nazarov cyclization reaction, Optical properties of carbon nanotubes, Saegusa–Ito oxidation, Weinreb ketone synthesis, and Wieferich prime. Best, Chemist234 (talk) 05:14, 16 March 2014 (UTC)

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GA Reassessment

Oligonucleotide synthesis

The following discussion is closed. Please do not modify it. Subsequent comments should be made on the appropriate discussion page. No further edits should be made to this discussion.

---

Article (edit | visual edit | history) · Article talk (edit | history) · Watch • Watch article reassessment page • GAN review not found

Result: Delisted. —Femke 🐦 (talk) 19:21, 19 February 2023 (UTC)

As per usual, some uncited material including

• The DMT group is removed with a solution of an acid, such as 2% trichloroacetic acid (TCA) or 3% dichloroacetic acid (DCA), in an inert solvent (dichloromethane or toluene). The orange-colored DMT cation formed is washed out; the step results in the solid support-bound oligonucleotide precursor bearing a free 5'-terminal hydroxyl group. It is worth remembering that conducting detritylation for an extended time or with stronger than recommended solutions of acids leads to depurination of solid support-bound oligonucleotide and thus reduces the yield of the desired full-length product.
• The reaction is also highly sensitive to the presence of water, particularly when dilute solutions of phosphoramidites are used, and is commonly carried out in anhydrous acetonitrile. Generally, the larger the scale of the synthesis, the lower the excess and the higher the concentration of the phosphoramidites is used. In contrast, the concentration of the activator is primarily determined by its solubility in acetonitrile and is irrespective of the scale of the synthesis. Upon the completion of the coupling, any unbound reagents and by-products are removed by washing.
• The First commercially available oligonucleotide synthesizers section (there's a weird external link in the middle of it)
• (2) The second approach is only used when the intended method of purification is reverse-phase HPLC. In this case, the 5'-terminal DMT group that serves as a hydrophobic handle for purification is kept on at the end of the synthesis. The oligonucleotide is deprotected under basic conditions as described above and, upon evaporation, is purified by reverse-phase HPLC. The collected material is then detritylated under aqueous acidic conditions. On small scale (less than 0.01–0.02 mmol), the treatment with 80% aqueous acetic acid for 15–30 min at room temperature is often used followed by evaporation of the reaction mixture to dryness in vacuo. Finally, the product is desalted as described above. For some applications, additional reporter groups may be attached to an oligonucleotide using a variety of post-synthetic procedures.

And more. These will have to be cited in order to remain a GA. Onegreatjoke (talk) 22:15, 7 February 2023 (UTC)

Pinging Chemist234 who nominated the article for GA in 2009. It's not clear if it got much of a GAN review at the time, so I'm not sure how much work would be needed to get it up to the criteria. But if C234 is still interested/able, perhaps they can deal with your notes above. Ajpolino (talk) 22:44, 7 February 2023 (UTC)

Just a few comments from someone who has not worked on this article and who is indebted to those that have done so much fine work on an important topic:

• • Statements like "...with a solution of an acid, such as 2% trichloroacetic acid (TCA) or 3% dichloroacetic acid (DCA), in an inert solvent (dichloromethane or toluene). The orange-colored DMT cation formed is washed out; the step results in the solid support-bound oligonucleotide precursor bearing a free 5'-terminal hydroxyl group." seems too detailed for encyclopedia.
  • Phrases like "It is worth remembering", "recommend" may be in conflict with WP:NOTMANUAL and/or WP:NOTTEXTBOOK.
  • Article is too heavy on primary references, many of which have been summarized in secondary references WP:SECONDARY and, in the case of this mature area, WP:TERTIARY. Large numbers of primary references put off readers and suggest the absence of any editor with an overview of the topic.
  • Article cites several patents. At least in Wikipedia:WikiProject Chemistry, we rarely cite patents. They are not peer-reviewed, they are often narrow. Most chem editors question whether they are RS.
  • Article uses the term "recent" several time. That adjective becomes stale with time.
  • Artcile uses the term "very" often. Such intensifiers lose their impact after multiple uses.
  • The section "Characterization" is sorta generic and not very interesting (to me at least).
  • --Smokefoot (talk) 23:39, 7 February 2023 (UTC)

The discussion above is closed. Please do not modify it. Subsequent comments should be made on the appropriate discussion page. No further edits should be made to this discussion.