Quantitative DNA methylation in DMRs of imprinted genes in 10.5dpc placentas as revealed by qAMP. Diabetic (n = 26 from 5 litters) and nondiabetic (n = 25 from 5 litters) placentas at mid-gestation were recovered. (A) The paternally methylated H19; (B, C) Percentage methylation values at single and groups of restriction sites in the maternally methylated DMRs of Snrpn and Peg3. The lines represent the DMRs which were analyzed and the letter indicates the recognition sites of the enzymes. M, McrBc; Hh, HhaI; Hp, HpaII. (D) Confirmation of gene methylation level was determined by qAMP to expected values. HhaI and McrBc sites in chr9:106724005–106724149 are unmethylated in the genome. A primer pair flanking these sites was utilized to amplify DNA (NIH 3 T3 mouse genomic DNA and CpG methylated NIH 3 T3 mouse genomic DNA, NEB) mixed at the ratios: 100:0, 75:25, 55:45, 50:50, 45:55, 0:100. The qAMP values were close to expected values. Data are presented as mean ± SE for each enzyme employed. White bar, non-diabetic group; black bar, diabetic group; *P < 0.05, **P < 0.01.