DNA synthesizer
From Biohack
(specifically, oligo arrayers)
Contents |
About the DNA synthesizer
DNA is the biochemical code that allows protein biosynthesis which basically leads to the entire construction of the organism less some bootstrapping problems (for example, how can you create the membrane from scratch when all cells except perhaps the first generation already had a plasma membrane). There are various ways of going about making new DNA sequences. The first one is to introduce very specific mutations by doing selection experiments and evolutionary engineering. Another method is making the DNA from scratch, or oligonucleotides.
The open source Piezoelectric Oligonucleotide Synthesizer And Microarrayer (POSAM Inkjet Microarrayer) (how to build it [pdf]) is a project that involves the hardware schematics and some software to control a DNA synthesizer. The design of this particular synthesizer involves using multiple servos to do horizontal and vertical positioning of a main 'bar'. This bar supports a number of cartridges which contain the chemicals required for the oligonucleotide reactions to construct DNA from scratch. Electric currents can signal the release of chemicals from particular cartridges once the bar has been adequately positioned. the positioning of the bar will usually correspond to the many (perhaps thousands) of wells on a metal surface below the bar. Once the synthesis is complete, each well will have the same strand (unless you are doing multiple syntheses at the same time) (redundancy is important - not all oligo-reactions work out perfectly (? Double check this.)).
Note that in the case of POSaM there is a piezoelectric unit that allows the 'ink' (chemicals) to be released for very brief moments, although the project description claims that typical inkjet cartridges from even Cannon or Epson can be used. Furthermore, POSaM has a chamber in which all of the reactions take place, so it has to be cased in glass and there has to be a vacuum (see Bryan's links re: vacuum chambers) and whatever remaining atmosphere has to be a gas (check the "how to build it" link (above)).
Ink cartridges in conventional printers
Most inkjet printers use cartdriges with colored dye. This dye is kept in the cartridge chamber, and a small internal micro jet turns on to activate the ink and project the dye on to the paper below it. There are 48 tiny holes in the bottom of the cartridge. Because of the heat and expansion/contraction of the ink within the cartridge, the ink bursts out of the bottom and splatters on the page in 48 separate (microscopic) dots.
Synthesis cycle
Oligonucleotide synthesis is done via a cycle of four chemical reactions that are repeated until all desired bases have been added:
- Step 1 - De-blocking (detritylation): The DMT is removed with an acid, such as TCA (get it at Sigma-Aldrich), and washed out, resulting in a free 5' hydroxyl group on the first base.
- Step 2 - Base condensation (coupling): A phosphoramidite nucleotide (or a mix) (struct, synthesis of phosphoramidite building blocks [pdf]) is activated by tetrazole (get) which removes the iPr2N group on the phosphate group. After addition, the deprotected 5' OH of the first base and the phosphate of the second base react to join the two bases together in a phosphite linkage. These reactions are not done in water but in tetrahydrofuran (get) or in DMSO (get). Unbound base and by-products are washed out.
- Step 3 - Capping: About 1% of the 5' OH groups do not react with the new base and need to be blocked from further reaction to prevent the synthesis of oligonucleotides with an internal base deletion. This is done by adding a protective group in the form of acetic anhydride (get) and 1-methylimidazole (get)which react with the free 5' OH groups via acetylation. Excess reagents are washed out.
- Step 4 - Oxidation: The phosphite linkage between the first and second base needs to be stabilized by making the phosphate group pentavalent. This is achieved by adding iodine (go to local store) and water which leads to the oxidation of the phosphite into phosphate. This step can be substituted with a sulphorylation step for thiophosphate nucleotides.
(Note: this might be a good document to see how phosphoramidites can be ordered from suppliers.) Here are some oligo synth protocols in molecbio. Quantifying oligos from phosphoramadite synth. Note that you may not have to actually purchase phosphoramadites to start off with, but instead begin with a purified solution of nucleic acid??
Oligo synth method over at John Hopkins Medical Institutes
The DNA syntheisis cycle begins with the 3'-hydroxyl nucleoside
attached to a solid Controlled-Pore Glass (CPG) support through a
long spacer arm. This support allows excess reagents to be removed by
filtration and eliminates the need for purification steps between
base additions.
The first step in the synthesis is the removal of the dimethoxytrityl
(DMT) group with TCA to free the 5'-hydroxyl for the coupling
reaction. The next step, activation, is achieved by simultaneously
adding a phosphoramidite derivative of the next nucleotide and
tetrazole, a weak acid to the reaction chamber. The tetrazole
protonates the nitrogen of the phosphoramidite, making it susceptible
to nucliophilic attack. This intermediate is very reactive and the
following coupling step is complete in less than 30 seconds. The
5'-OH group of the phosphoramidite is blocked with the DMT group.
The capping step terminates any chains that did not undergo coupling.
The unreacted chain has a free 5'-OH which can be terminated or
capped by acetylation to become "failure products". This is achieved
with acetic anhydride and 1-methylimidazole. The DMT group of the
successful coupling step protects the 5'-OH end from being capped.
Although this step is not absolutely necessary for DNA synthesis, it
minimizes the length of impurities and thus facilitates trityl-on
HPLC Purification.
The internucleotide linkage is then converted from the less stable
phosphite to the phosphotriester in the oxidation step. Iodine is
used as the oxidizing agent and water as the oxygen donor. (For
phosphorothioate oligonucleotides, the internucleotide phosphite is
sulfurized between coupling and capping).
After oxidation, the DMT group is removed with trichloroacetic acid
and the cycle is repeated until chain elongation is complete. The
oligo is then cleaved from the solid support with concentrated
ammonium hydroxide. Ammonia treatment also removes the cyanoethyl
phosphate protecting groups. The crude DNA in ammonium hydroxide
solution is then heated at 65 degrees for 1 hour to remove the
protecting groups on the exocyclic amines of the base.
This product is lyophilized and can be used as is or purified by
reverse-phase HPLC.
Some notes
Affymetrix uses photocleavage of the protecting groups on the dNTP monomers for their in situ synthesized oligonucleotide arrays, a sort of "photolithographic technology." Evidently they are using masks to write entire genomes in one go (just like in typical lithography techniques in the semiconductor industry), and so most of the time to synthesize DNA has transferred to making the masks. Quoteth: "(Xeotron and Nimblegen are now using dynamic micromirror displays for maskless photolithographic oligoarray synthesis, but these are also unavailable to most labs)".
The more nozzles, the faster your synthesis.
Quoting Wikipedia: "One technique used to produce oligonucleotide arrays include photolithographic synthesis (Agilent and Affymetrix) on a silica substrate where light and light-sensitive masking agents are used to "build" a sequence one nucleotide at a time across the entire array. [9] Each applicable probe is selectively "unmasked" prior to bathing the array in a solution of a single nucleotide, then a masking reaction takes place and the next set of probes are unmasked in preparation for a different nucleotide exposure. After many repetitions, the sequences of every probe become fully constructed. More recently, Maskless Array Synthesis from NimbleGen Systems has combined flexibility with large numbers of probes. [10]"
The NimbleGen maskless array synthesis is actually rather simple in its design and can be easily reverse-engineered with the content that they give on their website. Take a look at this diagram.
[Image:http://www.nimblegen.com/images/synthesis_illustration.jpg]
Essentially, it is as they say on their website: "At the heart of the system is a Digital Micromirror Device (DMD), similar to Texas Instruments' Digital Light Processor (DLP), employing a solid-state array of miniature aluminum mirrors to pattern 786,000 to 4.2 million individual pixels of light. The DMD creates "virtual masks" that replace the physical chromium masks used in traditional arrays. These "virtual masks" reflect the desired pattern of UV light with individually addressable aluminum mirrors controlled by the computer. The DMD controls the pattern of UV light projected on the microscope slide in the reaction chamber, which is coupled to the DNA synthesizer. The UV light selectively cleaves a UV-labile protecting group at the precise location where the next nucleotide will be coupled. The patterns are coordinated with the DNA synthesis chemistry in a parallel, combinatorial manner such that 385,000 to 2.1 million unique probe features are synthesized in a single array." (In this sense, array is kind of like a slide in a microscope, or even a wafer, on which works of art are built.)
Note that making such a micromirror system may be out of reach of the average individual, thus denying the possibility of combinatorial photolithographic oligonucleotide synthesis. The same photolithographic techniques can be employed on a much smaller scale with the POSaM setup.
"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)."
