PCR analysis of clonotypic genomic inv(16) (CBFB-MYHII)and t(15;17) (PML-RARA)sequences in neonatal Guthrie cards of leukemic patients. For analysis of Guthrie cards, 1/16 pieces were placed directly in first-round PCR reactions (50 μL), and for each individual, we tested at least 8 such segments (1/2 of a 1.5-cm spot). Second-round amplification (25 μL) was performed with 1 μL from the first-round PCR reactions. PCR conditions included 2 preincubation steps of 80°C for 15 minutes and 94°C for 4 minutes, followed by 40 amplification cycles of 94°C for 30 seconds, 58°C for 1 minute, 72°C for 1 minute, and, finally, a 72°C extension step for 7 minutes. Results shown are from the second round of nested PCR analysis. Lane markers: M indicates molecular weight marker. Lanes 1-5 show 1:10 dilutions of patient diagnostic DNA with lane 1 being 10 ng/μL and lane 5, 1 pg/μL DNA. G indicates patient Guthrie card samples; CC, control Guthrie card samples (from a healthy nonaffected individual); B, blank (no DNA sample). Arrows indicate PCR products from patient Guthrie segments. (A) CBFB-MYHII case no. P5: a single patient Guthrie segment yielded a PCR product from 6 analyzed. (B) PML-RARA case no. P1: 3 patient Guthrie segments yielded positive PCR products from 6 analyzed.