Biology Reference
In-Depth Information
photo-cleavage methods. The main advantage of this method is that it relies on
conventional phosphoramidite chemistry, without the cost or availability limitations
of photolabile protection groups. Photoacid generator (PAG) may liberate free radicals
to produce errors in synthesis. Serafinowski and Garland
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developed and used two
photosensitive esters, R-phenyl-4,5-dimethoxy-2-nitrobenzyltrichloroacetate and R-phenyl-
4,5-dimethoxy-2,6-dinitrobenzyltrichloroacetate, as PAG agents for synthesis to avoid
these problems.
Zhou et al.
11,36
coupled the PAG technique with a parallelized microfluidic platform.
An array chip was installed in a flow-through cartridge connected to a commercial
oligonucleotide synthesizer (Expedite 8909). The chips used differential surface tension
to isolate reaction sites, and allowed for the miniaturization of synthesis. Cleaved
oligonucleotides were of high enough quantities and qualities to be successfully assembled
in 10 kb constructs with PCR and ligation.
Electrochemical Arrays
Another method of DNA synthesis involves using localized electrochemical reactions
for the deblocking step. An array of platinum microelectrodes is covered with an electrolyte
solution. Current is simultaneously applied to individually addressable microelectrodes
with a semiconductor circuit, so that the electrolytes near activated anodes are oxidized and
release acid for deblocking. In one study, the electrolyte used was 25 mM hydroquinone
and 25 mM benzoquinone with 25 mM tetrabutylammoniumhexafluorophosphate in
anhydrous acetonitrile.
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By pairing each anode with adjacent cathodes which can reduce
acid, the produced acid is confined. The acid then diffuses to the layer of substrate which
contains the oligonucleotides being synthesized. The electrodes were made by thin-film
photolithography of 50-nm thick iridium metal and were durable enough to use over
500 cycles without deteriorations. The authors have demonstrated the synthesis of
short 17 bp oligonucleotides with complete electrochemical deblocking in as little as
9 seconds.
8
Customarray
s oligonucleotides arrays are also synthesized using a semiconductor-based
electrochemical synthesis process. Rather than using cathodes to localize generated acids,
synthesis is performed on a polymer surface over the surface of the semiconductor.
The porous polymer slows down acid diffusion to prevent cross-contamination between
local electrodes. The surface also increases the oligonucleotide density during synthesis.
Customarray
'
'
s 90 K microarrays contain 94 000 unique oligonucleotides and 25
μ
m
features, and are capable of synthesizing oligonucleotides of up to 50 bp lengths.
Besides feature density, another advantage of electrochemical synthesis is flexibility.
Because photolithographic masks are not involved, it is less expensive and requires less
labor to change the array design. Additionally, electrochemical detritylation is a relatively
efficient chemistry; the deblocking step can be complete in seconds and any side reactions
from the electrolyte chemicals are minimal. Efficient chemistry and a lack of moving parts
contribute to a total synthesis time of less than 24 hours.
Inkjet Printing
In 1981, Hood and Caruther modified liquid phase phosphite-triester chemistry for solid-
phase DNA synthesis on polymer supports.
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Solid-phase synthesis not only became the
method of choice for conventional DNA synthesis, but also opened DNA synthesis
to automated and miniaturized strategies on microarrays by eliminating the need to purify
synthetic intermediates or unreacted reagents. The first inkjet DNA synthesizers used
in-house microfabricated piezoelectric actuators.
8
Piezoelectric inkjet DNA synthesizers
operate similarly to commercial inkjet printers. Instead of ink, the inkjet head contains four
phosphoramidite fluid channels, one activator channel, and one optional modified base
