Figure 4.

Detection of the A13G mutant miR-128b allele in a primary MLL-AF4 ALL tumor. (A) Schematic of the procedure used to enrich the mutant alleles is shown along with the gel images that are analyzed at each step. After the 1^st genomic PCR, the target bands are seen in all samples. The arrow indicates the expected size of the PCR product. After digestion of the 1^st PCR products by Tsp45I, which cleaves the wild-type but not the A13G mutant DNA, no bands corresponding to the undigested PCR product, which corresponds to the mutant allele, are visible in any of these samples. The arrows (top to bottom) indicate the predicted sizes of DNAs corresponding to the undigested and the two digested products. After the 2^nd semi-nested PCR, all samples show the expected amplified species; the arrow indicates the expected size of the PCR product. 1–9 indicated patients 1 to 9; a–f, normal control a, b, c; P, positive control that contains 10% of mutant and 90% wild type DNA. (B) After the digestion of the 2^nd PCR products by Tsp45I, patients 1 and 5 show an undigested DNA, which corresponds to the mutant allele, as indicated by the upper arrows (upper left). Other patient samples show no undigested DNA (i.e., wild-type sequence; lower left). The DNA sequence analysis demonstrated that the PCR product from patient 5, as well as the positive control (P) show a G base at position 13, that is, the A13G mutation, while patient 1 showed only an A base at this position (right). The arrow indicates the position of A13G mutation.