Multiplexed, particle-based detection of DNA using flow cytometry with 3DNA dendrimers for signal amplification.
BACKGROUND
Complex mixtures of DNA may be found in environmental and medical samples. There is a need for techniques that can measure low concentrations of target DNAs. For a multiplexed, flow cytometric assay, we show that the signal-to-noise ratio for fluorescence detection may be increased with the use of 3DNA dendrimers. A single fluorescent DNA molecule per bead could be detected with conventional flow cytometry instrumentation.


METHODS
The analyte consisted of single-stranded (ss) DNA amplicons that were hybridized to capture probes on the surface of fluorescent polystyrene microspheres (beads) and initially labeled with streptavidin-R-phycoerythrin (single-step labeling). These beads have a low reporter fluorescence background and high efficiency of DNA hybridization. The DNA/SA-RPE complex was then labeled with 3DNA dendrimers and SA-RPE. The bead complexes were detected with a Luminex 100 flow cytometer. Bead standards were developed to convert the intensity to the number of SA-RPE labels per bead and the number of dendrimers per bead.


RESULTS
The dendrimer assay resulted in 10-fold fluorescence amplification compared with single-step SA-RPE labeling. Based on concentration curves of pure target ss-amplicons, the signal-to-noise ratio of the dendrimer assay was greater by a factor of 8.5 over single-step SA-RPE labeling. The dendrimer assay was tested on 16S ribosomal DNA amplified from filter retentates of contaminated groundwater. Multiplexed detection of a single dendrimer-labeled DNA molecule per bead was demonstrated.


CONCLUSIONS
Multiplexed detection of DNA hybridization on a single molecule level per bead was achieved with conventional flow cytometry instrumentation. This assay is useful for detecting target DNAs at low concentrations.
Lowe
Mary
M
Physics Department, Loyola College in Maryland, Baltimore, Maryland 21210, USA. mlowe@loyola.edu
Spiro
Alex
A
Zhang
Yu-Zhong
YZ
Getts
Robert
R
D002138:Calibration; D004247:DNA / Q000032:analysis* / Q000737:chemistry; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D008863:Microspheres; D009693:Nucleic Acid Hybridization; D010799:Phycoerythrin / Q000737:chemistry; D012680:Sensitivity and Specificity; D019809:Streptavidin / Q000737:chemistry
D005456:Fluorescent Dyes; D010799:Phycoerythrin; D004247:DNA; D019809:Streptavidin