Comparison of different labeling methods for the production of labeled target DNA for microarray hybridization.
Different labeling methods were studied to compare various approaches to the preparation of labeled target DNA for microarray experiments. The methods under investigation included a post-PCR labeling method using the Klenow fragment and a DecaLabel DNA labeling kit, the use of a Cy3-labeled forward primer in the PCR, generating either double-stranded or single-stranded PCR products, and the incorporation of Cy3-labeled dCTPs in the PCR. A microarray that had already been designed and used for the detection of microorganisms in compost was used in the study. PCR products from the organisms Burkholderia cepacia and Staphylococcus aureus were used in the comparison study, and the signals from the probes for these organisms analyzed. The highest signals were obtained when using the post-PCR labeling method, although with this method, more non-specific hybridizations were found. Single-stranded PCR products that had been labeled by the incorporation of a Cy3-labeled forward primer in the PCR were found to give the next highest signals upon hybridization for a majority of the tested probes, with less non-specific hybridizations. Hybridization with double-stranded PCR product labeled with a Cy3-labeled forward primer, or labeled by the incorporation of Cy3-labeled dCTPs resulted in acceptable signal to noise ratios for all probes except the UNIV 1389a and Burkholderia genus probes, both located toward the 3' end of the 16S rRNA gene. The comparison of the different DNA labeling methods revealed that labeling via the Cy3-forward primer approach is the most appropriate of the studied methods for the preparation of labeled target DNA for our purposes.
Franke-Whittle
Ingrid H
IH
Institute for Microbiology, University of Innsbruck, Technikerstrasse 25d, A-6020 Innsbruck, Austria. ingrid.whittle@uibk.ac.at
Klammer
Susanne H
SH
Mayrhofer
Sabine
S
Insam
Heribert
H
D016956:Burkholderia cepacia / Q000235:genetics*; D002232:Carbocyanines / Q000737:chemistry; D004256:DNA Polymerase I / Q000737:chemistry; D004269:DNA, Bacterial / Q000737:chemistry* / Q000235:genetics; D005456:Fluorescent Dyes / Q000737:chemistry; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016133:Polymerase Chain Reaction; D012336:RNA, Ribosomal, 16S / Q000737:chemistry* / Q000235:genetics; D013194:Staining and Labeling / Q000379:methods; D013211:Staphylococcus aureus / Q000235:genetics*
D002232:Carbocyanines; D004269:DNA, Bacterial; D005456:Fluorescent Dyes; D012336:RNA, Ribosomal, 16S; C089187:cyanine dye 3; D004256:DNA Polymerase I