Amplification of antigen receptor gene rearrangements and fusion regions of MLL-AF4/AF4-MLL rearrangements in neonatal blood spots from children with ALL using radioactive nested (TCRG, fusion sequences) or seminested (IgH, pt 3) or a nonradioactive seminested (IgH, pts 1 and 2) ASO-PCR, respectively.
Radioactive PCR products were size fractionated on denaturing polyacrylamide gels and exposed overnight. Amplified IgH products were separated on 3% agarose gels. Patients 1 and 2: (A) Amplification of the leukemia clone-specific IgH rearrangements (size: 75 base pairs (bp) and 74 bp for patients 1 and 2, respectively). (B) Amplification of the leukemia-specific MLL-AF4 (patient 1) and AF4-MLL (patient 2) fusion sequence resulting in a band of 146 bp and 143 bp, respectively. Patients 3, 4, and 5: Amplification of the leukemia clone-specific IgH rearrangement (67 bp in patient 3) and TCRG rearrangement (148 bp and 124 bp for patients 4 and 5, respectively). SM: Size marker VIII (Boehringer Mannheim, Mannheim, Germany); controls: lane 1: no DNA; lane 2: peripheral blood DNA; lanes 3-7: dilutions of leukemic DNA in DNA from peripheral blood mononuclear cells from healthy donors from 10−3 to 10−7; a sensitivity of 10−5 was achieved for all clonotypic rearrangements; lane 8: Guthrie card DNA; lane 9: Guthrie card DNA from age-matched controls.
