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 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.

The illusion of specific capture: surface and solution studies of suboptimal oligonucleotide hybridization

“…[20] Subsequently they were dried and scanned. The third QC assay was a TDT end labeling assay, in which one slide from each batch was covered with 1 uM cy3 dCTP (GE cat# PA 53021), 1× TDT reaction buffer and 1 unit dTDT (Affymetrix PN# 72033) [21] and incubated at 37°C for 25 min. Slides were washed twice with 0.1 × SSC and dried for scanning.…”

Terminal Deoxynucleotidyl Transferase in the Synthesis and Modification of Nucleic Acids

“…Indeed, TdT‐mediated polymerization reactions often produce long polydisperse tails that can complicate quantification of the labeled DNA. To circumvent this issue, base‐modified dideoxynucleotides (ddN*TPs) or ribonucleotides can be used instead of dN*TPs, as the fluorescent readout signal is then directly proportional to the amount of 3′‐OH termini present in the analyte . In this context, different nucleotide analogues have been evaluated for their capacity to fluorescently label arrays consisting of 1273 short oligonucleotides; of these, only Cy5‐UTP 5 (Scheme ) is consistently well accepted by the TdT without being affected by the nucleotide composition at the 3′‐termini of the sequences.…”

“…To circumvent this issue, base-modified dideoxynucleotides (ddN*TPs) or ribonucleotides can be used insteado fd N*TPs, as the fluorescent readout signal is then directly proportionalt ot he amounto f 3'-OH termini present in the analyte. [83] In this context, different nucleotide analogues have been evaluated for their capacity to fluorescently label arrays consisting of 1273 short oligonucleotides;o ft hese, only Cy5-UTP 5 (Scheme 1) is consistently well accepted by the TdT without being affected by the nucleotide composition at the 3'-termini of the sequences.A ll other ddN*TPs and N*TPs lead to lower labeling efficiencies if the primerc ontains a3 '-terminal dG residue. [18e, 40,83] Another important parameter that needs to be taken into consideration is the influence of the natureo ft he modification on the substrate acceptance of the triphosphateb yt he TdT.…”

“…[83] In this context, different nucleotide analogues have been evaluated for their capacity to fluorescently label arrays consisting of 1273 short oligonucleotides;o ft hese, only Cy5-UTP 5 (Scheme 1) is consistently well accepted by the TdT without being affected by the nucleotide composition at the 3'-termini of the sequences.A ll other ddN*TPs and N*TPs lead to lower labeling efficiencies if the primerc ontains a3 '-terminal dG residue. [18e, 40,83] Another important parameter that needs to be taken into consideration is the influence of the natureo ft he modification on the substrate acceptance of the triphosphateb yt he TdT. As ystematic investigation of fluorescently labeled dATPs and dUTPs has revealed that the polymerization reactioni sn ot very efficient if the modified triphosphates are used as the only monomer.O n the other hand, upon using Cy3-dATP/dATP,r atios of approximately 0.2 lead to the formation of 1kblong chains containing up to 50 fluorophores;t his efficiency is approximately 15 times superiort ot hat for the PCR-mediated incorporation of Cy3-dAMP.T he combinationo fn aturala nd modified nucleotides for the tail labeling of oligonucleotides with fluorophores has also been exploited to detect DNA hybridizatione ventsd irectly on chips.…”

Detection and Differentiation of Four Poultry Diseases Using Asymmetric Reverse Transcription Polymerase Chain Reaction in Combination with Oligonucleotide Microarrays

“…5 Virus isolation, antigen detection, and serologic analysis are the methods currently used for the laboratory diagnosis of these viruses. Viral isolation techniques are considered the gold standard for identification of active viral infections in blood and tissues.…”