Genetic variation detected by quantitative analysis of end-labeled genomic DNA fragments.

Abstract

The continuing efforts to evaluate specific human populations for altered germinal mutation rates would profit from more efficient and more specific approaches than those of the past. To this end, we have explored the potential usefulness oftwo-dimensional electrophoresis ofDNA fragments obtained from restriction-enzyme-digested genomic DNA. This permits the analysis, on a single preparation, of -2000 DNA fragments varying in size from 1.0 to 5.0 kb in the first dimension and from 0.3 to 2.0 kb in the second dimension. To enter into a genetic analysis, these fragments must exhibit positional and quantitative stability. With respect to the latter, if spots that are the product of two homologous DNA fragments are to be distnguished with the requisite accuracy from spots that are the product of only one fragment, the coefficient of variation of spot intensity should be approximately -0.12. At present, 482 of the spots in our preparations meet these standards. In an examination of preparations based on 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 cloning a variant fragment from such gels and establishing its nucleotide sequence. This technology should be highly efficient in monitoring for mutations resulting in loss/pin/rearrangement events in DNA fragments distributed throughout the genome.Research conducted in recent years has revealed a staggering wealth of genetic variation in the DNA of humans and other animals. Better techniques for the rapid identification and genetic analysis of this variation and for determining the frequency of germinal and somatic mutation, including the alterations in DNA associated with oncogenesis, are highly desirable. The advent of two-dimensional separations of genomic DNA fragments may be an important advance in this context (1)(2)(3)(4)(5). In this communication employing the technique of end-labeled restriction landmarks, we will describe the implementation of an approach for the quantitative analysis of the human DNA fragments visualized in autoradiographs of sheet gels prepared using two-dimensional electrophoresis. Elsewhere, we describe the qualitative variation detected with this technology (R.K., J.A., J.V.N., C.S., and S.M.H., unpublished data). Here, we emphasize the ability to distinguish between the autoradiographic intensity of a spot that is the product of two homologous DNA fragments as contrasted with the intensity of a fragment corresponding to one copy of the same DNA fragment. The ability to make this distinction with high accuracy provides the basis for employing this technique in the study of the frequency of mutation. Finally, we will consider the sources of nongenetic variation in spot density and discuss how the detection of genetic variation in gels of this type might be improved. MATERIALS AND METHODSDNA Samples. The DNA analyzed was obtained from three father/mother/one-child family constellat...

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