This paper describes a method for directly determining unique DNA sequences from mouse genomic DNA. The method involves denaturing gel electrophoresis of DNA fragments from complete restriction and partial chemical cleavages of the entire genome. These DNA fragments are transferred to nylon membranes and hybridized with a short, 32P-labeled single-stranded probe. This produces a DNA sequence "ladder" extending from the 3' or 5' end of a restriction site in the genome. The same membrane can be reprobed to obtain numerous different sequences. Each band in these sequences represents 3 fg of DNA complementary to the probe. The method is applicable to the analysis of genetic polymorphisms, DNA methylation at deoxycytidines, and nucleic acid-protein interactions at single nucleotide resolution. The method allows visualization of individual nucleotides within large chromosomes. During recombinant DNA cloning, information about DNA methylation and chromatin structure is lost. Direct chemical modification of the genome combined with complete restriction enzyme digestion and separation by size on a denaturing gel preserves some of this information in the form of numerous comigrating sets of DNA sequence "ladders." To access one sequence at a time, the lanes of DNA are transferred and crosslinked to a nylon membrane and hybridized to a short single-stranded 32P-labeled probe specific for one end of one restriction fragment within the genome. The probe called "3' lower" will hybridize to only three classes of DNA reaction products: the appropriate fragments extending from the 3' end of the lower strand of the restriction fragment, the longest fragments from the 5' end of the upper strand, and the middle fragments with both ends produced by chemical cleavage. The abundance of the appropriate fragments is proportional to the probability that the chemical reaction cleaves at any given target. Because the abundance of the middle fragments is proportional to P², interference can be diminished by decreasing the extent of the reaction. About one cleavage every 500 nucleotides is optimal. DNA methylation is discussed, with up to 12% of all cytosines in vertebrate genomes being methylated mainly at C-G sequences. In plants, up to 50% of all cytosines are methylated mainly at C-G and C-N-G sequences. Only a small subset of these methylation sites can be assayed by restriction analyses. The method allows quantitation of methylation levels of individual cytosine bases in DNA from various tissues. The same DNA replicated in Escherichia coli acts as an unmethylated control. The paper also describes the materials and methods used for DNA sequencing, including DNA samples, nylon membranes, electrophoretic transfer, UV irradiation, DNA probe synthesis, RNA probe synthesis, and hybridization. The results and discussion include an example of the methylation of cytosines in a region from the mouse IgM heavy chain constantThis paper describes a method for directly determining unique DNA sequences from mouse genomic DNA. The method involves denaturing gel electrophoresis of DNA fragments from complete restriction and partial chemical cleavages of the entire genome. These DNA fragments are transferred to nylon membranes and hybridized with a short, 32P-labeled single-stranded probe. This produces a DNA sequence "ladder" extending from the 3' or 5' end of a restriction site in the genome. The same membrane can be reprobed to obtain numerous different sequences. Each band in these sequences represents 3 fg of DNA complementary to the probe. The method is applicable to the analysis of genetic polymorphisms, DNA methylation at deoxycytidines, and nucleic acid-protein interactions at single nucleotide resolution. The method allows visualization of individual nucleotides within large chromosomes. During recombinant DNA cloning, information about DNA methylation and chromatin structure is lost. Direct chemical modification of the genome combined with complete restriction enzyme digestion and separation by size on a denaturing gel preserves some of this information in the form of numerous comigrating sets of DNA sequence "ladders." To access one sequence at a time, the lanes of DNA are transferred and crosslinked to a nylon membrane and hybridized to a short single-stranded 32P-labeled probe specific for one end of one restriction fragment within the genome. The probe called "3' lower" will hybridize to only three classes of DNA reaction products: the appropriate fragments extending from the 3' end of the lower strand of the restriction fragment, the longest fragments from the 5' end of the upper strand, and the middle fragments with both ends produced by chemical cleavage. The abundance of the appropriate fragments is proportional to the probability that the chemical reaction cleaves at any given target. Because the abundance of the middle fragments is proportional to P², interference can be diminished by decreasing the extent of the reaction. About one cleavage every 500 nucleotides is optimal. DNA methylation is discussed, with up to 12% of all cytosines in vertebrate genomes being methylated mainly at C-G sequences. In plants, up to 50% of all cytosines are methylated mainly at C-G and C-N-G sequences. Only a small subset of these methylation sites can be assayed by restriction analyses. The method allows quantitation of methylation levels of individual cytosine bases in DNA from various tissues. The same DNA replicated in Escherichia coli acts as an unmethylated control. The paper also describes the materials and methods used for DNA sequencing, including DNA samples, nylon membranes, electrophoretic transfer, UV irradiation, DNA probe synthesis, RNA probe synthesis, and hybridization. The results and discussion include an example of the methylation of cytosines in a region from the mouse IgM heavy chain constant