Nucleoside phosphoramidites are used to synthesise short nucleic acid chains. The chemical process allows several modifications, such as linker arms or using alternative nucleotides, such as LNA or morpholino or 2' group modified (OMe, NH2, F) or abasic, non-canon bases (xanthine, hypoxantine, triclycic bases etc) or bases with a fluorescent group, linker arm to attach a fluorescent group (aminoallyl)or biotin attached and so forth. There are a variety of alternative chemical and methods to do so, in fact Pubmed list nearly a thousand articles that modify this method . The prices depend on the company and the quantity required and are around 0.17$-0.30$ a base for DNA,while higher prices for RNA 3.50-4.50$/base (technology is not that effiecient)and other varaints (IDT , invitrogen ). The name nucleoside phosphoramidite comes from the phosphite group that has a NH2 instead of a OH group, the "phosphate" group in normal nucleic acids is pentavalent, while here it is trivalent. The structure of amidophosphoric acid is present in pubchem . The efficiency of the chemical synthesis is about 98% per base (see capping step). this is ideal for short oligos, but when 100 or more bases are made the method is not that good and enzymatic ligation is used. IUPAC nomenclature is a system of naming chemical compounds and of describing the science of chemistry in general. ...
A chemical formula (also called molecular formula) is a concise way of expressing information about the atoms that constitute a particular chemical compound. ...
The molecular mass (abbreviated MM) of a substance, formerly also called molecular weight and abbreviated as MW, is the mass of one molecule of that substance, relative to the unified atomic mass unit u (equal to 1/12 the mass of one atom of carbon-12). ...
CAS registry numbers are unique numerical identifiers for chemical compounds, polymers, biological sequences, mixtures and alloys. ...
Schematic diagram of a double-stranded nucleic acid. ...
Aminoallyl nucleotides are used in postlabelling of nucleic acids to be used in microarrays. ...
Solid phase chemical sythesis
It is done by protection chemistry were the most reactive groups are protected to avoid unwanted products. Whereas in biological processes it is nearly a dogma that enzymes work on DNA 5' to 3', this artificial chemical process is done backwards. 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 DMT (dimethoxytrityl) group. The free phosphoarmidite bases have on their phopshate group an 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 TBS (butyldimethylsilyl) group or with a Me group.
- Capping: The few (1%) free 5'OH must be stopped, their are capped with acetic anhydride and 1-methylimidazole.
- Oxidation: The phophate 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 hydrolisis (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). Aminoallyl nucleotides are used in postlabelling of nucleic acids to be used in microarrays. ...
T4 RNA ligase and T4 DNA ligase are used for making long oligos by ligating 2 short oligos (5'Phosphate donor and 3'OH acceptor). Template driven enzymatic sythesis is also effiencient when using T7 Polymerase or the Klenow fragment and modified bases.
An interesting development of this technology has allowed genechips to be made, where the probes are synthetised on the silicon chip, and not printed, allowing a higher resolution. This can be done via a mechanical mask were thin silicon rubber capillaries are put on a glass slide and the probes synthetised. 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 to generate microarray chips to have 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).
- primers for PCR and RT PCR
- 70mers for printer microrarrays
- 18mers in situ synthesis of probes for a genechip
Small interfering RNA (siRNA) are a class of 20-25 nucleotide-long RNA molecules that interfere with the expression of genes. ...
Primer can refer to more than one thing: In molecular biology, a primer is nucleic acid strand (or related molecule) that serves as a starting point for DNA replication. ...
Verma S, Eckstein F.1998 Modified oligonucleotides: synthesis and strategy for users. Annu Rev Biochem.67:99-134.
Brown T, Brown DJS. 1991. In Oligonucleotides and Analogues. A Practical Approach, ed. F Eckstein, pp. 1– 24. Oxford: IRL