Figure 4.

Assay for both NciI polymorphism and CfoI methylation at PW71 reveals loss of allelic methylation in peripheral blood and T cell clones. (A) Primers were designed to span both a polymorphic NciI site (∗) and a diagnostic site of allelic methylation (+). (B) Genomic DNA from a plasmid clone of the region (p1022) and peripheral blood from a normal individual heterozygous for the NciI site (NS2.PBL) were predigested with (+) or without (−) CfoI prior to PCR amplification. After amplification, PCR products were then digested with (+) or without (−) NciI to determine methylation status of each allele. NS2.PBL DNA predigested with CfoI showed a predominance of the NciI− allele, demonstrating partial allelic methylation. (C) Genomic DNA from peripheral blood (NS2.PBL), PBLs stimulated and cultured for around 25 generations with PHA (NS2.PHA), and two T cell clones (NS2.1 and 2) was assayed for allelic methylation as described in B. NS2.PHA and NS2.PBL exhibited the same preference for the methylated allele, but the two T cell clones varied in the degree of methylation of the NciI+ allele. CfoI predigestion was complete, as determined by the lack of amplification of a sequence containing unmethylated CfoI sites from the BRCA2 locus (data not shown).