The GNAS1 exon 1A region is methylated only on the maternal allele in normal subjects. (a) Genomic DNA from two normal subjects heterozygous for a 5-bp insertion polymorphism in GNAS1 exon 1A (N1 and N2) was digested with PstI and AscI, and fractions containing the 2.8-kb methylated (Meth) and 1.6-kb unmethylated (Unmeth) fragments were purified from agarose gels. The fractions were genotyped for the exon 1A polymorphism using PCR and direct sequencing and for parental assignment were run next to the same reaction performed on genomic DNA from a homozygous parent (mother for N1, father for N2). The presence of the polymorphic 5-bp insertion is indicated with brackets (although there is compression on these gels, bisulfite-modified genomic sequencing, shown in b, which removed these compressions, confirmed that the insertion is in fact 5-bp long). For both N1 and N2, the genotypes of methylated and unmethylated fragments corresponded to those of the mother and father, respectively. Identical results were also obtained for N3, the sister of N2 (data not shown). (b) Results of bisulfite-modified genomic sequencing of N3, showing the methylated maternal allele on the left and the unmethylated paternal allele (which includes the 5-bp insertion polymorphism) on the right. In the paternal allele, all Cs are unmethylated and are therefore converted to Ts. In the maternal allele all Cs within CpG dinucleotides (eight shown in figure; 25 total in sequencing reaction) are methylated and therefore remain as Cs (Cs that are not within CpG dinucleotides are unmethylated and are therefore converted to Ts).