Fluorescent ASO-specific PCR assay. RNA was extracted from a dilution of BaF3 cells expressing the T315I mutant of BCR-ABL in K562 cells, reversely transcribed into cDNA, and subjected to fluorescent ASO-specific PCR with primers optimized for detection of the T315I mutant. Plasmid DNA was included as a positive control. (A) Detection of amplification products by SybrGreen. 1 indicates plasmid DNA (control); 2 to 5, dilutions from 1:0 to 1:103; and 6, dilutions of 1:104 and 1:105 (curves overlap). (B) Melting curve analysis. Only selected melting curves are shown, and the colors do not correspond to panel A. 1 indicates curves corresponding to dilutions of 1:10, 1:103, and 1:105; 2, dilution of 1:106; and 3, K562 cells. (C) The PCR products of the fluorescent assay were run on a 1% agarose gel. Lane 1 indicates molecular weight marker; DNA, diluted plasmid DNA. Note the absence of PCR product in the 1:106 dilution and the K562-only specimen. The quality of cDNA was routinely checked by amplification of ABL as a control gene (not shown).
