Detection of NOTCH1 mutation via PCR. (A) Specificity of PCR primers for patient-specific mutations was examined by PCR with diagnostic DNA samples of T-ALL patients (MGT01 to MGT11). Primers used for the PCR are shown with the panels. Two rounds of PCR were necessary to obtain a clear amplification band in patient MGT09, possibly because of a low number of leukemic cells. (B) Sensitivity assay of mutation-specific primers. Serially diluted diagnostic DNA was subjected to PCR amplification with Ampdirect Plus in 40 cycles of PCR reaction. F indicates forward and r, reverse PCR primers for mut, mutation. Sequences of the primers for specific mutation of each patient were as follows: MGT01mut-r, 5′-GTGCGTCACGCTTGGCGACTTTTG-3′ for patient MGT01; MGT09mut-r, 5′-CGTCACGCTTGGGCACAAAG-3′ for MGT09 and MGT11mut-f; 5′-GAGAGCCGAGCCAGGCACACT-3′ for MGT11. MGT01mut-r and MGT09mut-r were used in combination with HDN-f, and MGT11mut-f was used with PEST-r primers. When necessary, 2-round, seminested PCR was conducted with the primers; HDN-f2, 5′-ACTGCGACCAGGGCTGCAACAG-3′ (with MGT01mut-r or MGT09mut-r) and PEST-r2, 5′-GTTGTCCACAGGCGAGGAGTAG-3′ (with MGT11mut-f). (C) Results of representative PCR for patient-specific mutation are shown. Of 8 MGT01 blood spot pieces, 5 showed positive bands (of which 2 were very faint and became clear after seminested PCR amplification (data not shown). All slices from blood spots of MGT09 and MGT11 were negative for their respective NOTCH1 mutation. At least 16 pieces of blood spots, in total, were examined in each patient. C indicates control blood spot without NOTCH1 mutation. (D) Results of SIL-TAL1 amplification on the MGT01 blood spot. Sixteen pieces were examined in total and all were negative, as shown here for 8 slices, even after 2 rounds of PCR amplification.