Digital sequence analysis of dna methylation

A procedure for selecting a defined subset of CpG loci in a marker nucleic acid for use in a nucleic acid detection assay that detects the coordinated methylation of a defined subset of CpG loci, wherein the coordinated methylation of the defined subset of CpG loci is indicative of adenoma or cancer, the procedure comprising: a) determining the methylation status of a plurality of CpG loci in each of a plurality of individual copies of a marker nucleic acid from a plurality of normal samples; b) determining the methylation status of said plurality of CpG loci in each of a plurality of individual copies of said marker nucleic acid from a plurality of non-normal samples from subjects having adenoma or cancer; wherein the plurality of individual copies of said marker nucleic acid analyzed in each of said normal samples and said adenoma samples or said cancer samples comprises at least 1,000; c) determining the methylation ratios of each CpG locus in said plurality of said CpG loci in said marker nucleic acid; wherein the determination of said methylation relationships comprises the determination of the relationship between the mean methylation in each of said plurality of CpG loci in said normal samples with respect to the mean methylation of each corresponding CpG locus in said plurality of loci CpG in such non-normal samples; d) selecting a defined set of CpG loci in said marker nucleic acid, wherein said defined set of CpG loci comprises at least three CpG loci having advantageous methylation ratios that correlate with adenoma or cancer; and e) selecting a defined subset of CpG loci from said defined set, wherein said defined subset of CpG loci comprises at least three CpG loci, and wherein said defined subset is selected such that the percentage of individual copies in said marker nucleic acid from said plurality of normal samples that are methylated at all said at least three CpG loci in said defined subset is less than the percentage of individual copies of said marker nucleic acid from said plurality of non-normal samples from subjects having adenoma or cancer that are methylated at all said at least three CpG loci in said defined subset.