Optimization of reverse hybridization in microplates coated with rRNA targeted oligonucleotide probes.
Among the modern molecular techniques for the identification of microorganisms the most straightforward way is through direct hybridization with rRNA/rDNA targeted probes. In this study, the optimization of the experimental procedures for the reverse hybridization technique in 96-well microplates is described using both synthetic model oligonucleotides (18 b) and amplified DNA (app. 4500 bp). Three different types of plates were compared (Maxi Sorp, NucleoLink, CovaLink). Plates made from nonchemically modified polystyrene which are conventionally used in immunoassays (MaxiSorp) proved to be an economic alternative for plates offering chemically modified tailor-made surfaces. Phosphorylation of the oligonucleotide probe was not necessary for successful immobilization whereas with 5'-terminal hexa-deoxyadenosine tailed capture oligonucleotides an enhanced sensitivity of the assay was observed. Variation of the stringency by adjusting different concentrations of formamide during the washing step ensures high probe specificity and therefore allows reliable identification of the microorganisms. The assay can be performed in less than 4 hours using pre-coated plates which can be stored for several weeks. After dissociation of the target DNA/capture probe duplex with an alkaline denaturing solution rehybridization is possible.
Institut für Wasserchemie, Technische Universität München, Germany.
M
M
Schedl
T
T
Behr
W
W
Ludwig
K H
KH
Schieifer
R
R
Niessner
D
D
Knopp
D015342:DNA Probes; D004275:DNA, Ribosomal / Q000032:analysis*; D016983:Enterococcus / Q000145:classification / Q000302:isolation & purification*; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D015347:RNA Probes; D012329:RNA, Bacterial / Q000235:genetics; D012335:RNA, Ribosomal / Q000032:analysis*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012680:Sensitivity and Specificity
D015342:DNA Probes; D004275:DNA, Ribosomal; D015345:Oligonucleotide Probes; D015347:RNA Probes; D012329:RNA, Bacterial; D012335:RNA, Ribosomal; D012336:RNA, Ribosomal, 16S