DIGITAL RESTRICTION ENZYME ANALYSIS OF METHYLATION

摘要

A method of analyzing the methylation of CpG sites where genomic DNA is sequentially digested with a pair of enzymes recognizing the same restriction site (CCCGGG) containing a CpG dinucleotide. The first enzyme, Smal, cuts only at unmethylated CpG and leaves blunt ends. The second enzyme, XmaI is not blocked by methylation and leaves a short 5' overhang. The enzymes thus create methylation specific signatures at ends of digested DNA fragments. These are deciphered by next generation sequencing. Methylation levels for each sequenced restriction site are calculated based on the numbers of DNA molecules with the methylated or unmethylated signatures. Using this method and by sequencing on a massively, parallel sequencing device, DNA methylation can be analyzed in a single blood sample.