A simple method for generating single-stranded DNA probes labeled to high activities.

The random priming DNA-labeling method of Feinberg and Vogelstein (1, 2) produces probes of high activities. However, incompletely denatured templates in the reaction mixture may cause problems. In addition, probes generated by the standard random priming method are not ideal for in situ hybridization or other methods requiring only one labeled strand. We have developed a method utilizing a biotinylated single-stranded template bound to magnetic microspheres in the standard random priming reaction. The template is generated by a PCR-reaction with one of the two primers biotinylated (Fig. 1). The biotinylated PCR-product is then bound to streptavidin-coated magnetic beads (Dynabeads M-280 Streptavidin, Dynal). The non-biotinylated strand is removed using alkaline treatment and magnetic separation (3). Labeling of the 'purified' biotinylated strand can then be carried out as a one-tube reaction since unincorporated nucleotides are removed by fixing the beads with the magnet and discarding the supernatant. The choice of biotinylated primer decides which strand is to be labeled. In contrast to probes generated by the M-13 system this method can use any vector that can serve as a PCR-template. Compared to RNA-probes the single-stranded DNA probes have the advantage that they are easier to handle; there is no need for enzymatic degradation of the template and contamination by RNase is no problem. The southern blot in Fig. 2, hybridized with a probe generated by this protocol (a) and with a conventional double-stranded probe (b), shows that the method generates a probe which specifically recognizes only one of the two complementary strands. The single-stranded probes described in this protocol give exposure times of 15-30 hours for genomic blots (8-10 /ig DNA per lane) for detection of single copy genes in barley DNA (data not shown). Protocol: PCR: 1 ng linearized DNA (450 bp insert of B19, a barley seed cDNA, in the vector pBluescript SK) in lxPCR-buffer (Promega), 200 mM dNTPs, 1 /tM each of T7 and SK primer (one of the two biotinylated), 2.5 U Taq polymerase (Promega), 30 cycles of 1 min/94°C, 2 min/45°C, 3 min/72°C (Temperature cycler; Techne PHC-2). The biotinylated PCR product (200-300 ng) was bound to 25 fd of magnetic streptavidin beads by incubation in 6XSSC for 2 min. Beads were washed once in 6XSSC and the non-biotinylated strand was removed by two cycles of denaturation in 100 /J of 0.125 M NaOH in 0.1 M NaCl. After washing twice with 1XSSC, 0.1 % SDS and twice …