High sensitivity mapping of methylated cytosines

TLDR

DNA methylation’s role in development, aging, and cancer is poorly understood because existing methods cannot resolve exact methylation patterns from small DNA amounts. The study seeks to establish procedures that maximize denaturation, bisulphite conversion, and amplification to map methylation of single genes from minimal genomic DNA. The method converts unmodified cytosines to uracil with sodium bisulphite while leaving 5‑methylcytosine intact, then amplifies the converted DNA with specific primers and sequences it, so remaining cytosines reveal methylated sites. The resulting sequencing technique detects every methylated cytosine on both strands of any target sequence using DNA from fewer than 100 cells.

Abstract

An understanding of DNA methylation and its potential role in gene control during development, aging and cancer has been hampered by a lack of sensitive methods which can resolve exact methylation patterns from only small quantities of DNA. We have now developed a genomic sequencing technique which is capable of detecting every methylated cytosine on both strands of any target sequence, using DNA isolated from fewer than 100 cells. In this method, sodium bisulphite is used to convert cytosine residues to uracil residues in single-stranded DNA, under conditions whereby 5-methylcytosine remains non-reactive. The converted DNA is amplified with specific primers and sequenced. All the cytosine residues remaining in the sequence represent previously methylated cytosines in the genome. The work described has defined procedures that maximise the efficiency of denaturetion, bisulphite conversion and amplification, to permit methylation mapping of single genes from small amounts of genomic DNA, readily available from germ cells and early developmental stages.