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. 2013 Jun 17;41(16):7700–7712. doi: 10.1093/nar/gkt524

Figure 2.

Figure 2.

Design and synthesis of strand-specific fluorescent probes for comet-FISH. (a) The 3′ and 5′ regions of the ATM gene are amplified by PCR using biotinylated forward primers, natural reverse primers and aminoallyl-dUTP. Subsequently, the biotinylated strands and the non-biotinylated strands of the PCR products are separated by streptavidin-coated beads. The probes targeting the 3′ and 5′ regions of the ATM gene are labeled with Alexa 488 and Alexa 594, respectively. Finally, all the biotinylated probes are combined as probes for the ATM TS, whereas all the non-biotinylated probes are mixed as probes for the ATM NTS. (b) Agarose gel containing the double-stranded PCR product (lane 1), single-stranded DNA with biotin (lane 2), single-stranded DNA without biotin (lane 3) and annealed double-stranded DNA (lane 4). (c) Agarose gel with fluorescently labeled strand-specific probes stained with ethidium bromide (left) or unstained (right). Probes for the 3′ region of the ATM TS (lanes 1 and 5), probes for the 3′ region of the ATM NTS (lanes 2 and 6), probes for the 5′ region of ATM TS (lanes 3 and 7), probes for the 5′ region of the ATM NTS (lanes 4 and 8). (d) Absorption spectra of probes targeting the 3′ region (green) and the 5′ region (red) of the ATM TS. (e) Absorption spectra of probes targeting the 3′ region (green) and the 5′ region (red) of the ATM NTS. The peaks centered at 260, 492 and 588 nm correspond to DNA, Alexa 488 and Alexa 594, respectively.