GENETIC-VARIATION DETECTED BY QUANTITATIVE-ANALYSIS OF END-LABELED GENOMIC DNA FRAGMENTS

The continuing efforts to evaluate specific human populations for alte red germinal mutation rates would profit from more efficient and more specific approaches than those of the past. To this end, we have explo red the potential usefulness of two-dimensional electrophoresis of DNA fragments obtained from restriction-enzyme-digested genomic DNA. This permits the analysis, on a single preparation, of approximate to 2000 DNA fragments varying in size from 1.0 to 5.0 kb in the first dimensi on and from 0.3 to 2.0 kb in the second dimension. To enter into a gen etic analysis, these fragments must exhibit positional and quantitativ e stability. With respect to the latter, if spots that are the product of two homologous DNA fragments are to be distinguished with the requ isite accuracy from spots that are the product of only one fragment, t he coefficient of variation of spot intensity should be approximately less than or equal to 0.12. At present, 482 of the spots in our prepar ations meet these standards. In an examination of preparations based o n three Japanese mother/father/child trios, 43 of these 482 spots were found to exhibit variation that segregated within families according to Mendelian principles. We have established the feasibility of clonin g a variant fragment from such gels and establishing its nucleotide se quence. This technology should be highly efficient in monitoring for m utations resulting in loss/gain/rearrangement events in DNA fragments distributed throughout the genome.