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. 2010 Jul-Sep;1(1):45–53. doi: 10.4161/adna.1.1.12444

Figure 4.

Figure 4

Purification of linear dsDNA containing the PNA-complementary sequence by affinity capture. (A–C) A mixture of three DNA fragments (lane mix) containing either the perfectly matched target (F-PM), single mismatch target SM-1 (F-SM), or no target sequence (F-NT) was incubated under different conditions with PNA1 (1.5 µM). Following removal of excess PNA, samples were incubated with streptavidin-coupled magnetic beads for 1 h at ambient temperature followed by collection of supernatant (lanes SN, uncaptured DNA fraction). Captured DNA was released by incubation of sample at 60°C in buffer containing 500 mM NaCl (lanes MB). Conditions of PNA1 binding: (A) 1 h at 42°C with 15 mM NaCl added; (B) 2 h at 42°C with 30 mM NaCl added; (C) 4 h at 42°C at with 45 mM NaCl added. M: 1 kb DNA ladder. (D) Capture of a short, perfectly matched target (PM) in the presence of human genomic DNA. Samples were analyzed on 8% native PAGE. Incubation with PNA1 was carried out for 4 h at 42°C with 35 mM NaCl. Lane Con is a control sample lacking incubation with beads. Lanes W1–W4 are samples obtained at washing of beads with buffer containing 0 mM, 5 mM, 50 mM and 100 mM NaCl, respectively, following the collection of supernatant. PM, unbound fragment; PM + PNA, strand-invasion complex. M: 2-log DNA ladder.