Here are some notes on oligonucleotide synthesis using phosphoramidite chemistry, including notes on an inkjet DNA synthesizer based on the [POSAM project](http://bioinformatics.org/pogo/).
# Summary The project goal is to design, build and operate an open-source DNA synthesizer. There are [many interesting DNA synthesis projects](http://diyhpl.us/wiki/dna/projects) that could be pursued given the availability of large quantities of cheap large-scale highly-parallel DNA synthesis. Ultimately the chemistry of phosphoramidite synthesis will guide and inform the machine design, but so far the best direction seems to be using an inkjet for depositing 100s of millions of drops per second of chemical reagents onto a surface. Each drop is a "pixel" that acts as a unique reaction chamber for the reagents. Each layer of the image builds upon the surface-bound reaction results of the previously deposited reagents. Different DNA molecules are formed inside each droplet pixel region on the surface. The DNA molecules are covalently bonded to either the surface or to microbeads on the surface. The droplets don't intersect or merge their contents together because of surface tension and distance. The inkjet can target each drop and deposit different chemical reagents into every drop on every "round" of the synthesis cycle. Because of side-reactions, moisture and other errors, the length of the DNA molecule that can be manufactured inside of each location is limited to perhaps 100 to 200 bp at most, and usually must be at least 20 bp to be of any utility. Conjugation and ligation is necessary to combine short DNA molecules into longer DNA molecules. Although the synthesis cycle is well-known, the design for mechanically moving the separate strands together remains to be determined for this DNA synthesis machine. Once short DNA molecules are placed together, reactions such as Gibson assembly or Ligase Cycling Reaction or extension PCR can be used to combine short DNA molecules into longer DNA molecules. The POSAM synthesizer was designed to create DNA hybridization arrays, where the DNA would be left on the surface and not combined into longer DNA molecules. Perhaps the machine's design will use really tiny pipette tips to transport short DNA from multiple drops to a single reaction chamber? The conjugation of these DNA molecules is critical to the formation of superlong DNA molecules between 5K bp (basepairs) and 1M base pairs. The human genome has approximately 3B base pairs, and DNA synthesizers from the 80s were making 100 bp molecules at best. Note that DNA synthesis and DNA sequencing are two distinct procedures. Sequencing is reading, synthesis is writing. The cost of DNA sequencing (reading) has been falling quickly over the past decade, but synthesis has not moved as much. The project is self-funded and communication with the team can be achieved by looking at the [[hplusroadmap]] IRC channel.
# Oligonucleotide synthesis using the phosphoramidite method ## Synthesis cycle steps The following steps are for the addition of each nucleotide to the growing oligos. It's not the complete set of steps. Each addition of a nucleotide requires the following steps. ### 1. Deblocking (detritylation) Detritylation occurs using an acid such as 2% [trichloroacetic acid (TCA)](https://en.wikipedia.org/wiki/Trichloroacetic_acid) or 3% [dichloroacetic acid (DCA)](https://en.wikipedia.org/wiki/Dichloroacetic_acid), in an inert solvent ([dichloromethane](https://en.wikipedia.org/wiki/Dichloromethane) or [toluene](https://en.wikipedia.org/wiki/Toluene)). - produces an orange-colored DMT cation which needs to be washed out - oligonucleotide precursor has a free 5'-terminal hydroxyl group - using a stronger acid or conducting detritylation for an extended time, leads to [depurination](https://en.wikipedia.org/wiki/Depurination) of the oligonucleotide ### 2. Coupling A 0.02–0.2 M solution of nucleoside phosphoramidite (or a mixture of several phosphoramidites) in acetonitrile is activated by a 0.2–0.7 M solution of an acidic azole catalyst (below). The mixing is usually very brief and occurs in fluid lines of oligonucleotide synthesizers (see below) while the components are being delivered to the reactors containing solid support. The extension reaction forms a phosphite triester linkage (below). 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.
acidic azole catalysts: - 1H-tetrazole - 2-ethylthiotetrazole - 2-benzylthiotetrazole - 4,5-benzylthiotetrazole - 4,5-dicyanoimidazole - alternatives can be found here: Wei, Xia (2013). "Coupling activators for the oligonucleotide synthesis via phosphoramidite approach". Tetrahedron 69 (18): 3615–3637. doi:[10.1016/j.tet.2013.03.001](https://dx.doi.org/10.1016%2Fj.tet.2013.03.001). phosphite triester linkage: The activated phosphoramidite in 1.5 – 20-fold excess over the support-bound material is then brought in contact with the starting solid support (first coupling) or a support-bound oligonucleotide precursor (following couplings) whose 5'-hydroxy group reacts with the activated phosphoramidite moiety of the incoming nucleoside phosphoramidite to form a phosphite triester linkage. Highly sensitive to water. Commonly carried out in anhydrous acetonitrile. ### 3. Capping - mixture of acetic anhydride and [1-methylimidazole](https://en.wikipedia.org/wiki/1-methylimidazole) (or less often, [DMAP](https://en.wikipedia.org/wiki/4-Dimethylaminopyridine)) as catalysts - unreacted 5'-hydroxy groups are acetylated by the capping mixture - capping reagent solution helps prevent reactions that might cleave the oligonucleotide (because of depurination, the apurinic sites are readily cleaved under the basic conditions of later steps). Capping reagent solution helps as long as the capping step is performed prior to oxidation with I2/water. ### 4. Oxidation The newly formed tricoordinated phosphite triester linkage is not natural and is of limited stability under the conditions of oligonucleotide synthesis. The treatment of the support-bound material with iodine and water in the presence of a weak base (pyridine, lutidine, or collidine) oxidizes the phosphite triester into a tetracoordinated phosphate triester, a protected precursor of the naturally occurring phosphate diester internucleosidic linkage. Oxidation may be carried out under anhydrous conditions using tert-Butyl hydroperoxide or, more efficiently, (1S)-(+)-(10-camphorsulfonyl)-oxaziridine (CSO). The step of oxidation is substituted with a sulfurization step to obtain oligonucleotide phosphorothioates (see Oligonucleotide phosphorothioates and their synthesis below). In the latter case, the sulfurization step is best carried out prior to capping. # solid supports # linker chemistry linkers go here # Inkjet printing steps 1. Printing 2. Washing 3. Oxidation 4. Washing 5. Detritylation 6. Washing # Reagent suppliers
# ABI 391 - [ABI 391 manual](http://diyhpl.us/~bryan/papers2/DNA/abi391/ABI%20391-manual.pdf) - [ABI 391 phosphoramidite chemistry from the manual](http://diyhpl.us/~bryan/papers2/DNA/abi391/chemistry.txt) - [chemical storage conditions](http://diyhpl.us/~bryan/papers2/DNA/abi391/Chemical%20Storage%20Conditions.pdf) - [replacing bottle seals on reagent bottles](http://diyhpl.us/~bryan/papers2/DNA/abi391/Replacing%20Bottle%20Seals%20on%20Reagent%20Bottles.pdf) - [takeitapart.com ABI 391 DNA synthesizer teardown](https://takeitapart.com/guide/94)
## ABI 391 reagents bottles: * bottle 1: adenosine phosphoramidite (A) (deoxyadenosine (dA-bz) phosphoramidite) (beta-cyanoethyl, as all the other phosphoramidites) * bottle 2: guanosine phosphoramidite (G) (deoxyguanosine (dG-ib) phosphoramidite) * bottle 3: cytosine phosphoramidite (C) (deoxycytosine (dC-bz) phosphoramidite) * bottle 4: thymidine phosphoramidite (T) (deoxythymidine (dT) phosphoramidite) * bottle 5: spare phosphoramidite reservoir for modified bases (X), such as deoxyinosine * bottle 9: tetrazole/acetonitrile (180 mL) * bottle 11: acetic anhydride/iutidine/THF (180 mL) * bottle 12: 1-methylimidazole (180 mL) * bottle 14: trichloroacetic acid (450 mL) * bottle 15: iodine/water/pyridine/THF (200 mL) * bottle 18: acetonitrile (4 L) also the following two might either be separate bottles or the same as bottle 18 (dunno): * anhydrous acetonitrile, for dissolving phosphoramidites * ammonium hydroxide also: * argon * concentrated ammonia * concentrated ammonium hydroxide * waste bottle (because "The synthesizer generates 1 to 2 liters of hazardous, halogenated, organic liquid waste per 100 base additions. The waste is collected in a 4 liter polyethylene bottle which is placed on the floor or on a nearby bench lower than the instrument. The bottle can be kept inside a protective carrier to contain accidental spillage. A 1 gallon carrier is sufficient and can be purchased. When the bottle is emptied, it must be emptied using a specific procedure.) There are three types of protecting groups: 1. 5' protecting group - dimethoxytrityl (DMT) 2. beta-cyanoethyl phosphate protecting groups 3. base protecting groups - benzoyl on dA and dC and isobutyryl on dG The 5'-protecting group, dimethoxytrityl, is attached to each nucleoside phosphoramidite and is cleaved from the growing oligonucleotide chain during each cycle of base addition. You can program the 391 to remove the last DMT to yield a 5' hydroxyl by choosing the ending method Trityl off, or leave the DMT on by choosing the ending method Trityl on. When purifying by polyacrylamide gel electrophoresis or ion exchange HPLC2, the last DMT group should be removed. When purifying by trityl-specific OPC or reverse phase HPLC, the last DMT group should be left on. To remove oligonucleotides from the solid supports, use concentrated ammonium hydroxide for simultaneous decyanoethylation and cleaving. Next, the base protecting groups are removed by the addition of fresh concentrated ammonia and incubation at 55 degrees celsius.
# POSAM * [posam user manual chapter 1: assembly](http://bioinformatics.org/pogo/POSAM_Man_Ch1_Assembly_v1-2_040601.pdf) * [posam user manual chapter 2: operations](http://bioinformatics.org/pogo/POSAM_Man_Ch2_User_v1-0_040414.pdf)
## Materials consumed per one inkjet oligonucleotide array from [table 1](http://www.ncbi.nlm.nih.gov/pmc/articles/PMC507883/table/T1/) slide derivatization: - glass slide - nano-strip glass cleaner - sodium hydroxide - hydrochloric acid - methanol - epoxysilane - rain-x silane solution - isopropanol - acetone chemical synthesis: - nitrogen - acetonitrile - 0.02 M iodine THF/Pyr/H2O - 2.5% DCA in DCM - dA-CE phosphoramidite - Ac-dC-CE phosphoramidite - dG-CE phosphoramidite - dT-CE phosphoramidite - tetrazole - ammonia - methylamine - ethanol - 3-methoxypropionitrile - 2-methyl glutaronitrile test hybridization: - lifterslip coverslips - DIG Easy-Hyb solution - control oligo (bodipy) - wash buffers miscellaneous materials: - septa - drierite - syringe, 1 mL - syringe, 5 mL - 26G needles - molecular sieves - inkjet head - exhaust filter cartridge - parafilm