Figure 5

Methylation analysis of individual blastocysts by bisulfite conversion followed by direct sequencing and cloning/sequencing. For each analyzed blastocyst, bisulfite mutagenesis was performed. After PCR amplification the methylation status of CpG positions was determined by direct sequencing (Figure 5A) and cloning/sequencing (Figure 5B). When Single Nucleotide Polymorphism (C/T) was observed by direct sequencing, the proportion of C in the clone sequences was approximately 50%. When only C or T were detected by direct sequencing, all sequences of analyzed clones presented a methylated or an unmethylated status respectively. Reading the direct sequences, for each blastocyst, the presence of C/T, C or T at one CpG position is represented by the black (methylated) and white (unmethylated) lollipops (Figure 5C).