QUANTITATIVE AND QUALITATIVE GENETIC-VARIATION IN 2-DIMENSIONAL DNA GELS OF HUMAN LYMPHOCYTOID CELL-LINES

There is a continuing need for more efficient methods to examine human (and other) populations for altered germinal and somatic cell mutatio n rates. To this end, we have explored the potential usefulness of two -dimensional (2-D) electrophoresis of human DNA fragments obtained fro m restriction-enzyme-digested genomic DNA, using samples from father/m other/child trios. On a single 2-D DNA preparation, approximately 2000 DNA fragments varying in size from 1.0 to 5.0 kbp in the first dimens ion and 0.3 to 2.0 kbp in the second dimension are visualized. To ente r into a genetic analysis of quantitative variation, these fragments m ust exhibit positional and quantitative stability. With respect to the latter, if spots that are the product of two homologous DNA fragments are to be distinguished with the requisite accuracy from spots that a re the product of only one fragment, the coefficient of variation of s pot intensity should be approximately less than or equal to 0.12. At p resent, 482 of the spots in our preparations meet these standards. In an examination of preparations based on three Japanese mother/father/c hild trios, 43 of these 482 spots were found to exhibit variations tha t segregated within families according to Mendelian principles. Additi onally, of the 2000 spots, 1114 (of which the aforementioned 482 are a subset) were deemed appropriate for the study of qualitative variatio n. A total of 142 variable spots were identified; the heterozygosity i ndex for these DNA fragments was 4.4%. The genetic nature of the addit ional variants was again established by their segregation according to Mendelian principles. We have established the feasibility of cloning fragments from such gels and determining their nucleotide sequence. Th is technology should be highly efficient in monitoring for mutation re sulting in loss/gain/rearrangement events in DNA fragments distributed throughout the genome.