Analysis of oligonucleotide microarrays by 3′ end labeling using fluorescent nucleotides and terminal transferase

Abstract

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, Cy™3-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.