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.
Physics Department, Loyola College in Maryland, Baltimore, Maryland 21210, USA. mlowe@loyola.edu
Mary
M
Lowe
Alex
A
Spiro
Yu-Zhong
YZ
Zhang
Robert
R
Getts
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