Talk:Oligonucleotide synthesis
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[edit] 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==
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==
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
[edit] 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)
