Recent highlights on photolithic oligonucleotide array in situ synthesis.
Light directed synthesis of high-density oligonucleotide microarrays is currently performed using either ortho-nitro-benzyl-type [MeNPOC] (Pease, A.C.; Solas, D.; Sullivan, E.J.; Cronin, T.M.; Holmes, C.P.; Fodor, S.P.A. Proc. Natl. Acad Sci U.SA. 1994, 91, 6333.) or ortho-nitrophenylethyl-type [NPPOC] (Hasan, A.; Stengele, K.P.; Giegrich, H.; Cornwell, P.; Isham, K.R.; Sachleben, R.A.; Pfleiderer, W.; Foote, R.S. Tetrahedron 1997, 53, 424Z) protecting groups as the 5'-O-carbonate ester of the phosphoramidite building block. The synthesis cycle uses a combinatorial approach attaching one specific base per cycle, thus as many as 100 cycles need to be run to make an array of 25-mers. Time needed for deprotection/activation of the growing oligo chain determines overall manufacturing time and consequently also cost. In this report we demonstrate the development of photoprotected posphoramidite monomers for light directed array synthesis with increasing sensitivity to the UV light used. If combined with maskless array synthesis, this technology allows for synthesis of arrays with >780,000 different 25-mer oligonucleotides in about one hour and allows for high flexibility in array design and reiterative redesign. The arrays synthesized show high quality and reproducibility in our standard hybridization based assay.
Stengele
Klaus-Peter
KP
Chemogenix GmbH, Waldkraiburg, Germany.
Bühler
Jochen
J
Bühler
Sigrid
S
Kvassiouk
Evgueni
E
Green
Roland
R
Prykota
Tamara
T
Pfleiderer
Wolfgang
W
D020650:Combinatorial Chemistry Techniques; D020869:Gene Expression Profiling / Q000295:instrumentation; D007700:Kinetics; D008956:Models, Chemical; D008967:Molecular Biology / Q000379:methods*; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation*; D015345:Oligonucleotide Probes; D009841:Oligonucleotides / Q000737:chemistry; D010777:Photochemistry; D020005:Propanols / Q000737:chemistry; D013997:Time Factors; D014466:Ultraviolet Rays
D015345:Oligonucleotide Probes; D009841:Oligonucleotides; D020005:Propanols