Nucleic Acids Research, 1990, Vol. 18, No. 4 809-815
© 1990
GENOME STRUCTURE AND MAPPING |
Detection of mutations and DNA polymorphisms using whole genome Southern Cross hybridization
1Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology Cambridge, MA 02139 2Department of Cellular and Developmental Biology, Harvard University Cambridge, MA 02138, USA
*To whom correspondence shonld be addressed at Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA
Received November 14, 1989. Accepted January 10, 1990.
We report a general method for the detection of restriction fragment length alterations associated with mutations or polymorphisms using whole genomic DNA rather than specific cloned DNA probes. We utilized a modified Southern Cross hybridizatIon to display the hybridization pattern of all size-separated restriction fragments from wild-type Caenorhabditls elegans to all the corresponding fragments in a particular mutant strain and in a distinct C. elegans variety. In this analysis, almost all homologous restriction fragments are the same size in both strains and result In an intense diagonal of hybridization, whereas homologous fragments that differ in size between the two strains generate an off-diagonal spot. To attenuate the contribution of repeated sequences in the genome to spurious off-diagonal spots, restriction fragments from each genome were partially resected with a 3' or 5' exonuclease and not denatured, so that only the DNA sequences at the ends of these fragments could hybridize. Off-diagonal hybridization spots were detected at the expected locations when genomic DNA from wild-type was compared to an unc-54 mutant strain containing a 1.5 kb deletion or to a C. elegans variety that contains dispersed transposon insertions. We suggest that this modified Southern Cross hybridization technique could be used to identify restriction fragment length alterations associated with mutations or genome rearrangements in organisms with DNA complexities as large as 108 base pairs and, using rare-cutting enzymes and pulse-field gel electrophoresis, perhaps as large as mammalian genomes. This information could be used to clone fragments associated with such DNA alterations.