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

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• 作者：Kathrin Br眉ckner ; Kathleen Schwarz ; Sebastian Beck ; Michael W. Linscheid
• 刊名：Analytical Chemistry
• 出版年：2014
• 出版时间：January 7, 2014
• 年：2014
• 卷：86
• 期：1
• 页码：585-591
• 全文大小：311K
• ISSN：1520-6882

文摘

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鈥搒treptavidin 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.