Analysis of oligonucleotide microarrays by 3' end labeling using fluorescent nucleotides and terminal transferase.
A simple enzymatic labeling procedure is described to determine spot quality in oligonucleotide microarrays. By using fluorescently labeled dideoxynucleotides or ribonucleotides as substrate for terminal deoxynucleotidyl transferase (TdT), a single fluorophore can be covalently attached at the 3' end of each oligonucleotide probe molecule in the spot. Fluorescein-12-ddUTP CyTM3-ddUTP Cy5-UTP, and Cy3-UTP were compared as TdT substrates for 3' end labeling an array of 1273 hexamer probes. Cy5-UTP was found to show minimal bias toward probe base composition and is therefore well suited for quantitative analysis of microarray spots where the oligonucleotide probes are coupled via a 5' end linkage to the solid phase.
Guerra
Cesar E
CE
Agilix Corporation, Woodbridge, CT 06525, USA. cguerra502002@yahoo.com
D004253:DNA Nucleotidylexotransferase / Q000235:genetics*; D015342:DNA Probes / Q000235:genetics; D005456:Fluorescent Dyes / Q000494:pharmacology*; D020869:Gene Expression Profiling; D009693:Nucleic Acid Hybridization; D009711:Nucleotides / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation* / Q000379:methods*; D015345:Oligonucleotide Probes / Q000235:genetics; D009841:Oligonucleotides
D015342:DNA Probes; D005456:Fluorescent Dyes; D009711:Nucleotides; D015345:Oligonucleotide Probes; D009841:Oligonucleotides; D004253:DNA Nucleotidylexotransferase