A simple non-isotopic method for DNA fingerprinting with M13 phage.

Recently it was discovered that the bacteriophage M13 contains in its protein III gene, tandem repeats of a 15-bp motif that can recognize a family of hypervariable minisatellites in vertebrate DNA (1). Southern blots of human DNA digested with Hae III and probed with 32p_iaDeled M13 revealed complex individual-specific DNA fingerprints, similar to those described by Jeffreys et al (2) with probes derived from the human myoglobin gene. We describe a simple non-isotopic method for DNA fingerprinting using biotinylated M13 phage as a probe. The method has resolution and sensitivity comparable to regular radioactive procedures. Moreover, it has the advantages of being safer and quicker, and the labeled probes can be stored for a long time and be reused many times. M13 LABELING: 17-mer universal M13 primer was hydridized to single stranded M13mpl8 phage and labeling was done by the incorporation of biotinyl-11-dUTP mediated by the Klenow fragment of DNA polymerase I, at room temperature (RT) for 15 h. No dTTP was included in the reaction mixture. In this fashion we achieved synthesis of a heavily biotinylated probe made up of the entire (-) strand of M13mpl8. Probes made by nick translation or by extension of random primers did not have the required sensitivity. SOUTHERN BLOTTING AND HYBRIDIZATION: 2-10 ng of human DNA were digested with Bsp RI (an isoschizomer of Hae III), electrophoresed in 0.8% agarose and blotted onto nylon or nitrocellulose membranes. The membranes were baked at 80°C for 1 h and then blocked for 2 h at 50°C in a solution containing 1% casein, 3% fish skin gelatin, 0.05% Tween 20, 0.5M NaCl and 0.1M Tris-HCl pH 7.5. Hybridization was done for 40 h at 42°C in a solution containing 45% formamide, 10% dextran sulfate, 5 X SSC, 0.1% SDS, 20mM sodium phosphate pH 7.0 and 1 X modified Denhardt's (fish skin gelatin substituted for BSA) as described by Dykes et al (3). Following hybridization, the membranes were washed 2 X 5 min at RT with 2 X SSC, 2 X 5 min at RT in 1 X SSC and 1 X 30 min with 1 X SSC at 50°C. They were then reblocked as before at RT and reacted with a streptavidin-biotinyl-alkaiine phosphatase complex (Enzo Biochem) diluted 1:500 in a 0.1M Tris-HCl pH 7.5 buffer containing 5% Tween 20 and 0.5M NaCl, for 1 h at room temperature. Finally the membranes were washed and color developed in a solution of 200 U g/ml BCIP and 50 u g/ml NBT overnight at room temperature. A representative result is shown in the figure. This successful development of a non-isotopic method for DNA fingerprinting with M13 represents a considerable simplification in methodology and is likely to increase its availability for paternity testing and forensic applications. This work is the subject of a patent application.