DNA quantification via ICP-MS using lanthanide-labeled probes and ligation-mediated amplification.
The combination of lanthanide-tagged oligonucleotide probes with inductively coupled plasma mass spectrometry (ICP-MS) as the detection technique is a novel labeling and analysis strategy for heterogeneous nucleic acid quantification assays. We describe a hybridization assay based on biotin-streptavidin affinity using lanthanide-labeled reporter probes and biotinylated capture probes. For the basic sandwich type assay, performed in streptavidin-coated microtitration wells, the limit of detection (LOD) was 7.2 fmol of DNA target, corresponding to a final concentration of 6 pM terbium-labeled probes detectable by ICP-MS after elution from the solid support. To improve the sensitivity and sequence specificity of the approach, it was combined with established molecular biological techniques, i.e., elution with a restriction endonuclease and signal and target amplification by the ligase detection reaction (LDR) and ligase chain reaction (LCR), respectively. Initial experiments showed that the enzymes facilitated the discrimination of single-base mismatches within the recognition or ligation site. Furthermore, LCR as a target amplification step resulted in a 6000-fold increase of sensitivity, and finally an LOD of 2.6 amol was achieved with an artificial double-stranded DNA target.
Department of Chemistry, Humboldt-Universitaet zu Berlin , Brook-Taylor-Strasse 2, 12489 Berlin, Germany.
Kathrin
K
Brückner
Kathleen
K
Schwarz
Sebastian
S
Beck
Michael W
MW
Linscheid
D000818:Animals; D002417:Cattle; D004247:DNA / Q000032:analysis* / Q000235:genetics; D011088:DNA Ligases / Q000032:analysis / Q000235:genetics; D028581:Lanthanoid Series Elements / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D009693:Nucleic Acid Hybridization / Q000379:methods*; D015345:Oligonucleotide Probes; D012710:Serum Albumin, Bovine / Q000032:analysis / Q000235:genetics; D013054:Spectrophotometry, Atomic / Q000379:methods*
D028581:Lanthanoid Series Elements; D015345:Oligonucleotide Probes; D012710:Serum Albumin, Bovine; D004247:DNA; D011088:DNA Ligases