Talk:Oligonucleotide synthesis

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Good article Oligonucleotide synthesis has been listed as one of the Natural sciences good articles under the good article criteria. If you can improve it further, please do so. If it no longer meets these criteria, you can reassess it.
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similarity[edit]

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 (talkcontribs) 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[edit]

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[edit]

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[edit]

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[edit]

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[edit]

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[edit]

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[edit]

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[edit]

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[edit]

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 pKA of Guanine is the highest with 12.3... Thet619 (talk) 23:29, 13 January 2013 (UTC)

  • Since we are discussing oligonucleotides, we should consider pKa of nucleosides or nucleotides rather than nucleic bases. For dG, there are several possible ionic equilibria. Thus, pKa of 12.3 refers to dissociation of hydroxy groups in 2'-deoxyribose. Further, pKa 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 pKa of N-protonated deoxyribonucleosides dG, dA, and dC is 2.4, 3.8, and 4.3, respectively. The pKa of N-protonated ribonucleosides G, A, and C is 2.1, 3.6, and 4.1, respectively. In ribonucleoside-5'-phosphates, pKa 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 pKa-values of the exocyclic amino groups of nucleosides/nucleotides? I only found the values of the exocyclic groups of nucleic bases (pKa 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 pKa for guanine is irrelevant here. I hope it helps, Chemist234 (talk) 19:59, 30 January 2013 (UTC)

Too Technical?[edit]

@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)