Analysis of DNA-microarrays produced by inverse in situ oligonucleotide synthesis.
5'-Phosphoramidites protected by 2-nitrophenylethyl (NPE) and 2-(4-nitrophenyl)ethoxy carbonyl (NPEOC) functions were employed for in situ synthesis of oligonucleotides in 5'-->3' direction on flat glass surfaces. By this inverse synthesis format, the oligonucleotides are attached to the solid support via their 5'-ends while the free 3'-hydroxyl groups are available as substrates for enzymatic reactions such as elongation by polymerases, thereby adding another feature to the portfolio of chip-based applications. Having a fluorescence dye present at the first base during synthesis, the quality of the oligonucleotides was analysed quantitatively by capillary electrophoresis after release from the solid support. With about 95% yield per condensation, it was found to be equivalent to synthesis results achieved on CPG support. The chip-bound oligonucleotides could be extended enzymatically upon hybridisation of a DNA-template. Surprisingly, however, only 63% of the oligonucleotides were elongated in polymerase reactions, while oligonucleotides that were released from the support behaved normally in standard PCR amplifications. This rate of 63% nevertheless compares favourably with an extension rate of only 50%, which was achieved under identical conditions, if pre-fabricated oligonucleotides of identical sequence had been spotted to the glass support.
Beier
Markus
M
Functional Genome Analysis, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 506, D-69120, Heidelberg, Germany. markus.beier@febit.de
Hoheisel
Jörg D
JD
D001483:Base Sequence; D001709:Biotechnology; D005456:Fluorescent Dyes; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis; D015345:Oligonucleotide Probes / Q000138:chemical synthesis / Q000737:chemistry; D016133:Polymerase Chain Reaction
D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes