Improved method for polynucleotide probe-based cell sorting, using DNA-coated microplates.
We developed an improved method for cultivation-independent sorting of bacterial cells. The technique is based on labeling the target cells by in situ hybridization with polynucleotide transcript probes. Due to the probes' length, part of the probe remains outside the cell and can subsequently be used to capture the cells. Target cells are immobilized during a second hybridization step in microplates that are coated with DNA that is complementary to the probe sequence. The method was applied successfully to artificial mixtures of cells with polynucleotide probes targeting either rRNA, a plasmid-borne beta-lactamase gene, or a chromosome-borne glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. Cells could be separated based on phylogenetic parameters (using rRNA-targeted probes) as well as on other DNA-encoded traits.
Zwirglmaier
K
K
Lehrstuhl für Mikrobiologie, Technische Universität München, 85350 Freising, Germany.
Ludwig
W
W
Schleifer
K-H
KH
D001431:Bacteriological Techniques; D018914:Cells, Immobilized; D004269:DNA, Bacterial / Q000032:analysis*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006090:Gram-Negative Bacteria / Q000166:cytology* / Q000201:enzymology / Q000235:genetics; D017403:In Situ Hybridization; D015345:Oligonucleotide Probes; D010957:Plasmids / Q000235:genetics; D011119:Polynucleotides; D012335:RNA, Ribosomal / Q000235:genetics; D001618:beta-Lactamases / Q000235:genetics
D004269:DNA, Bacterial; D015345:Oligonucleotide Probes; D011119:Polynucleotides; D012335:RNA, Ribosomal; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D001618:beta-Lactamases