Highly specific and sensitive detection of leukemia- and clone-specific genetic targets. Numbers and initials refer to the respective patient identification in Table 1. (A) Typical examples of an albumin RQ-PCR from Guthrie card DNA indicating the range of variability (translating to approximately 100 to 1000 cells per sample). External DNA standard at dilutions 100 ng, 10 ng, and 1 ng are in gray; Guthrie card DNA, black. (B) Representative example for quantification of the preleukemic/leukemic clone by allele-specific RQ-PCR of TCR rearrangements. Curves represent 10-log dilutions of leukemic DNA into PB starting from 10^-2 to 10^-5 in duplicates depicted in different shades of gray. Background amplification is shown by light gray dotted lines; no specific signal from Guthrie card DNA is detectable. (C-E) Detection of TAL1 deletions (C), TCRD-LMO2 breakpoint regions, (D) and Notch1 mutation (E) by a nested PCR approach. (C) Polyacrylamide gels showing second-round PCR products of TAL1 deletions; S, size marker; lanes 1 to 6, 10-log dilutions of leukemic DNA in PB from 10^-1 to 10^-6; lane 7, PB DNA; lane 8, no DNA; A, aliquots of Guthrie card DNA from the particular patient; C, control Guthrie cards. A vertical line has been inserted to indicate where a gel lane was cut. These gels came from the same experiments. (D,E) Polyacrylamide gel electrophoresis of second-round PCR products of TCRD-LMO2 breakpoints (D) and Notch1 mutation (E); lane 1, 10^-4 dilutions of leukemic DNA in PB; lanes 2 to 5, 10^-5 dilutions; lane 6, 10^-6 dilution; lane 7, PB DNA; lane 8, no DNA; A and C, as before; T, thymus DNA.