DNA Quantification via ICP-MS Using Lanthanide-Labeled Probes and Ligation-Mediated Amplification

• Published in: Analytical chemistry (Washington) 2014-01, Vol.86 (1), p.585-591
• Main Authors: Brückner, Kathrin, Schwarz, Kathleen, Beck, Sebastian, Linscheid, Michael W
• Format: Article
• Language: English
• Subjects: Amplification; Analytical chemistry; Animals; Assaying; Biological; Cattle; Chain reactions; Chemical elements; Deoxyribonucleic acid; DNA; DNA - analysis; DNA - genetics; DNA Ligases - analysis; DNA Ligases - genetics; Elution; Enzymes; Hybridization; Lanthanoid Series Elements - chemistry; Mass spectrometry; Mass Spectrometry - methods; Nucleic Acid Hybridization - methods; Oligonucleotide Probes; Serum Albumin, Bovine - analysis; Serum Albumin, Bovine - genetics; Spectrophotometry, Atomic - methods
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac402668p

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.