Method for examination of DNA to determine the presence of 5-methylcytosine based on the specific reaction of bisulfite (bisulphite) with cytosine

摘要

A method for the characterizing, classifying and differentiating tissues and cell types to predicate the behaviour of tissues and groups of cells and to identify genes with modified expression comprises treating genomic DNA which has been obtained from any sample and which may have been treated by being subjected to shearing or cleaved by means of a restriction endonuclease, with a bisulfite solution which converts the base cytosine, but not 5-methylcytosine into uracil. Fractions of the treated DNA are amplified using nonspecific hybridizing oligonucleotides, a mixture of two or more different specific hybridizing oligonucleotides and /or oligonucleotides which are complementary to adaptor oligonucleotides that have been ligated to the end of the cleaved DNA before the bisulfite treatment. The quantity of the remaining cytosine on the guanine-rich DNA strand and/or guanines on the cytosine-rich DNA strand from the amplified fractions are detected by hybridization or polymerase reaction which is such the data generated in such an analysis and automatically applied to a processing algorithm make it possible to draw conclusions regarding the phenotype of the analysed cell material. The method can further comprise after several test on DNA samples from phenotypically identical or similar cells or tissue correlating the data obtained with the phenotype of the cell whose DNA was examined in a, training phase using a neural network or evaluation algorithm. The data included in the training phase are used in the connection between the phenotype and the methylation state to derive by the generation of a methylation state of a DNA sample of unknown origin the phenotype of the cells whose DNA was examined and the data on the pattern on the methylation state of the DNA of a known cell type are used for identifying cytosine positions which differ in the examined DNA from the methylation state determined in the training phase.