jo970872s_si_001.pdf (102.58 kB)
Proofing of Photolithographic DNA Synthesis with 3‘,5‘-Dimethoxybenzoinyloxycarbonyl-Protected Deoxynucleoside Phosphoramidites
journal contribution
posted on 1998-01-01, 00:00 authored by Michael C. Pirrung, Lara Fallon, Glenn McGallWe have evaluated in a microchip format the photochemical
solid-phase phosphoramidite DNA
synthesis method we previously developed. A set of nucleoside
building blocks with “easy-off” base
protecting groups was prepared bearing photolabile
5‘-O-dimethoxybenzoincarbonate (DMBOC)
groups. Photolysis rates and cycle yields for these
DMBOC-protected nucleotides covalently attached
to planar, derivatized glass surfaces were determined by fluorescence
imaging-based methods earlier
developed by McGall et al. and described in detail elsewhere. Data were
obtained for both 280/310
and 365/400 nm irradiation in a range of solvents. Deprotection of
the DMBOC occurs fastest in
a nonpolar medium or without solvent. The coupling efficiency of
these amidites in the synthesis
of homopolymers was determined to be in the range 80−97%, with
purines generally showing lower
efficiency than pyrimidines. These DMBOC-protected monomers were
used to prepare a 4 × 4
array of 16 decanucleotides of the sequence 5‘-AAXTAXCTAC−chip, where
X = A, C, G, or T. The
array was hybridized with a target deoxyeicosanucleotide of the
sequence fluorescein-5‘-CTGAACGGTAGCATCTTGAC. Surface fluorescence imaging demonstrated
sequence-specific hybridization
to this probe.
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Keywords
probeGTAGCATCTThybridizationnonpolar mediumhomopolymerstarget deoxyeicosanucleotideProofingirradiationsequencemethodsolventefficiencyamiditemonomerPhotolithographic DNA Synthesisnucleoside building blocksphosphoramiditecycle yieldsfluoresceinpyrimidinearraypurinePhosphoramiditesynthesismicrochip formathybridizedcovalentlyphotolabilealPhotolysis ratesnmDeprotectionMcGallGACderivatized glass surfacesdataDMBOCAAXTAXCTACDeoxynucleoside16 decanucleotidesSurface fluorescence imaging
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