Two-dimensional DNA fingerprinting of human individuals

ABSTRACT The limiting factor in the presently available techniques for the detection of DNA sequence variation in the human genome is the low resolution of Southern blot analysis. To increase the analytical power of this technique, we applied size fractionation of genomic DNA restriction fragments in conjunction with their sequence-dependent separation in denaturing gradient gels; the two-dimensional separation patterns obtained were subsequently transferred to nylon membranes. Hybridization analysis using minisatellite core sequences as probes resulted in two-dimensional genomic DNA fingerprints with a resolution of up to 625 separated spots per probe per human individual; by conventional Southern blot analysis, only 20-30 bands can be resolved. Using the twodimensional DNA fingerprinting technique, we demonstrate in a small human pedigree the simultaneous transmission of 37 polymorphic fragments (out of 365 spots) for probe 33.15 and 105 polymorphic fragments (out of 625 spots) for probe 33.6. In addition, a mutation was detected in this pedigree by probe 33.6. We anticipate that this method will be of great use in studies aimed at (i) measuring human mutation frequencies, (ii) associating genetic variation with disease, (iii) analyzing genomic instability in relation to cancer and aging, and (iv) linkage analysis and mapping of disease genes. The possibility to identify DNA sequence heterogeneity is of major importance for the analysis of genetic diseases and genomic instabilities. This identification depends on the availability of probes that detect variable sites in the genome and on the resolution of electrophoretic separation techniques for the analysis of DNA restriction fragments. The discovery of hyperpolymorphic VNTR (variable number of tandem repeat) DNA sequences or minisatellites has greatly facilitated studies of genetic variation in the human population (1-6). It has been demonstrated that so-called core probes derived from minisatellites can be used to simultaneously detect a large number of hyperpolymorphic VNTR loci, dispersed in the genome, to provide genetic fingerprints of human individuals Gels were stained for 10 min in a solution containing ethidium bromide (EtdBr) at 0.1 ug/ml followed by destaining for at least 30 min. Two-dimensional separations of 5 ,ug of DNA restriction fragments were performed in 1-mm-thick polyacrylamide gels (acrylamide/bisacrylamide, 37:1) using a gel apparatus that was essentially the same as the one described by Fischer and Lerman (12). The first dimension was run in a neutral 6% gel at 50'C for 2 hr at 250 V in lx TAE. The separation patterns were visualized by staining the gel in the dark with EtdBr (0.1 ,ug/ml) for 10 min, followed by destaining for at least 30 min. The 0.34-to 2.8-kbp region (probe 33.15) or the 0.54-to 10-kbp region (probe 33.6) of the lane was quickly cut out of the gel and applied to a 6% polyacrylamide gel containing a 10-75% linear concentration gradient of denaturant (100% denaturant = 7.0 M urea/40% formamide) parallel to the direction of electrophoresis. This gradient was found to give optimal separation patterns for the VNTR sequences. Gels were poured by mixing two solutions, containing the desired boundary denaturant concentrations, in a linear gradient maker with a peristaltic pump. After electrophoresis for 12 hr at 225 V and 60'C, the gel was stained with EtdBr as described above. Transfer of Separation Patterns. DNA separation patterns in agarose gels were capillary transferred to a nylon membrane (Nytran 13N, Schleicher & Schuell; Zetaprobe, BioRad) in 0.4 M NaOH/0.6 M NaCl for 12 hr. After transfer, the filter was rinsed in 2x SSC (lx SSC = 150 mM NaCI/15 mM Abbreviations: EtdBr, ethidium bromide; VNTR, variable number of tandem repeats. 2742 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact