Methylation status of

p15^INK4b andp14^ARF analyzed by MSP. DNA was phenol/chloroform extracted from blood. Promoter methylation was determined by the method of MSP.4 The modified DNA was used as a template for PCR amplification using primers specific for either methylated or unmethylated DNA forp14^ARF,7,p15^INK4b, andp16^INK4a.4 The specificity of the PCR reaction was checked by sequencing the amplified fragments. Control without DNA was performed for each set of PCRs. Placental DNA treated or not in vitro with SssI methyltransferase (New England Biolabs, Beverly, MA) was used as positive control for the methylated or the unmethylated form, respectively. Examples are given for patients 9 to 19. M indicates methylated form; U, unmethylated form; P, unmethylated placental DNA; and SssI, methylated placental DNA. The DNA standards (methylated or unmethylated) gave the expected results.