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GO-based removal of PCR primer and sequence-specific amplicon detection.

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posted on 2017-08-29, 17:37 authored by Anna Maria Giuliodori, Anna Brandi, Shivaram Kotla, Francesco Perrozzi, Roberto Gunnella, Luca Ottaviano, Roberto Spurio, Attilio Fabbretti

(A) The DNA samples prepared as described in the text were incubated with the amounts of GO indicated below. After centrifugation, an aliquot of each supernatant was subjected to 1.5% agarose gel electrophoresis followed by ethidium bromide staining and band visualization under UV light. Orange and green bands correspond to cspC DNA and fluorescent primer, respectively. Lanes 1–2: no GO; lanes 3–4: 0.018 mg/ml GO; lanes 5–6: 0.032 mg/ml GO; lanes 7–8: 0.05 mg/ml GO; lane 9: 0.068 mg/ml GO; lane 10: 0.086 mg/ml GO (B) Quantification by densitometry of the bands shown in (A); black circles: cspC DNA; red squares: fluorescent primer. The percentage was calculated taking the average band intensities of lanes 1 and 2 as 100%. (C) Relative Fluorescence emitted by the samples incubated with the indicated amounts of GO (blue) or by the supernatants of the same samples recovered after centrifugation at 10 K rpm (pink) or 2 K rpm (green). (D) FAM-P (75 nM) was incubated with the indicated Target DNAs in the presence of Taq Buffer, dNTPs and Taq DNA Polymerase at the concentrations indicated in Materials and Methods. Fluorescence was measured after GO addition.