Combining the preparation of oligonucleotide arrays and synthesis of high-quality primers.
Based on the oligomer-chip technology, oligonucleotide arrays were synthesized directly on polypropylene sheets by a modified phosphoramidite chemistry using beta-eliminating nucleobase-protecting groups in combination with a succinate solid-phase linker. This method decouples the oligonucleotide deprotection from the support cleavage procedure, in contrast to standard phosphoramidite chemistry. In addition to being reliable substrates for hybridization experiments, the arrays also serve as source for the isolation of individual oligonucleotides. Technically, this allowed for a direct control of the quality of the arrayed oligomers. The released compounds were sufficient in amount and purity to work without further purification in PCR and DNA-sequencing reactions, with the results being identical to controls with commercially obtained primer molecules. Consequences for oligomer-chip hybridization procedures, the applicability of such hybrid-function arrays in, for example, diagnostics or comparative biology, and developments toward parallel primer synthesis are discussed.
Weiler
J
J
Molecular-Genetic Genome Analysis, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 506, Heidelberg, D-69120, Germany. j.weiler@dkfz-heidelberg.de
Hoheisel
J D
JD
D001483:Base Sequence; D001671:Biochemistry / Q000295:instrumentation / Q000379:methods / Q000592:standards; D017931:DNA Primers / Q000138:chemical synthesis* / Q000235:genetics / Q000302:isolation & purification*; D008722:Methods; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D011126:Polypropylenes; D011786:Quality Control; D017422:Sequence Analysis, DNA; D013499:Surface Properties
D017931:DNA Primers; D015345:Oligonucleotide Probes; D011126:Polypropylenes