Biomedical Engineering Reference
In-Depth Information
hv
Mask
T
O
O
O
O O
OHOH
Mask protecting
group MeNPOC
O
O
O
Substrate
(i)
(ii)
hv
C
TT
C
C
TT
TT
O O
O
O
O
O
O
(iii)
(iv)
(v)
C
A
T
A
T
A
G
C
T
G
TT
CC
G
(vi)
FIGURE 11.2 Photolithographic synthesis of oligonucleotide probe arrays.
(i) Glass substrate coated with silane coupling agent is illuminated through a lithography mask with many
segments of blocked and unblocked regions;
(ii) Areas under the unblocked segment of the mask are activated, resulting in the addition of nucleoside
phosphoramidite monomers;
(iii) The phosphoramidite group reacts with the hydroxyl group on the substrate in the presence of the silica
coupling agent;
(iv) A new mask is used and the surface is illuminated once again;
(v) A new nucleoside phosphoramidite monomers is coupled. Procedure is repeated as many times as need
to achieve the required length of the nucleotide chain (usually 25-mer or less).
Source: www.affymetrix.com
In DNA array fabrication by photolithography, a fused silica substrate is coated with
a silane coupling agent with a hydroxyl functional group in order to serve as the initial
synthesis site. In a report by Pease et al. [10], this substrate reacts with the DMT-hexa-
ethyloxy-O-cyanoethyl phosphoramidite linker. This linker is protected with a protec-
tive photolabile group (
-methyl-2-nitropiperonyl oxycarbonyl) (MeNPOC) which is
activated when regions of the surface are exposed to ultraviolet or deep ultraviolet light,
resulting in the addition of nucleoside phosphoramidite monomers. The phosphoramidite
group reacts with the hydroxyl group on the substrate in the presence of the silica cou-
pling agent. Under the UV radiation, the photolabile MeNPOC group produces the ø OH
group. The next MeNPOC protected nucleotide is added and coupled to the free ø OH
group of the grafted molecule. The MeNPOC group protecting the 5
α
end of the added
nucleotide is removed by UV radiation and this procedure is repeated as many times as
required to achieve the required length of the nucleotide chain (usually 25-mer or less).
The photolithographic synthesis of oligonucleotide probe arrays is shown in Fig. 11.2.
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