Open AccessAnalysis of oligonucleotide microarrays by 3′ end labeling using fluorescent nucleotides and terminal transferase
Author(s) -
Cesar E. Guerra
Publication year - 2006
Publication title -
biotechniques/biotechniques
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.617
H-Index - 131
eISSN - 1940-9818
pISSN - 0736-6205
DOI - 10.2144/000112182
Subject(s) - terminal deoxynucleotidyl transferase , oligonucleotide , fluorophore , nucleotide , microbiology and biotechnology , transferase , random hexamer , dna microarray , substrate (aquarium) , oligomer restriction , chemistry , biology , fluorescein , biochemistry , fluorescence , enzyme , dna , tunel assay , gene expression , gene , apoptosis , ecology , physics , quantum mechanics
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.