Methylation-specific digital karyotyping.
Epigenetic alterations, including DNA methylation and histone modifications, are known to regulate various physiological and pathological processes. In mammalian cells, DNA methylation occurs at cytosines of CpG dinucleotides. Several methods have been developed for the genome-wide analysis of methylation patterns. However, none of these are quantitative or sequence-based, and the identification of the exact location of the methylated CpG is difficult. In this protocol, we describe a recently developed method--methylation-specific digital karyotyping (MSDK)--that enables comprehensive and unbiased genome-wide DNA methylation analysis. Using a combination of a methylation-sensitive mapping enzyme (for example, AscI) and a fragmenting enzyme (for example, NlaIII), short sequence tags can be obtained and uniquely mapped to genome location. The number of tags in an MSDK library reflects the methylation status of the mapping enzyme sites. Generation of MSDK libraries can be completed in 7-10 days, whereas sequencing and data analysis requires an additional 3-4 weeks.
Hu
Min
M
Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA.
Yao
Jun
J
Polyak
Kornelia
K
D019175:DNA Methylation; D015252:Deoxyribonucleases, Type II Site-Specific; D044127:Epigenesis, Genetic; D015723:Gene Library; D023281:Genomics / Q000379:methods*; D007621:Karyotyping / Q000379:methods*
C078382:endodeoxyribonuclease AscI; C048422:endodeoxyribonuclease NlaIII; D015252:Deoxyribonucleases, Type II Site-Specific