Myeloid Leukemia

102 Branford and Hughes
Fig. 2. Determination of the sensitivity of mutation analysis. The sensitivity of the
mutation analysis was determined by preparing a dilution series of a BCR-ABL-expressing
cell line that contained the Y253F mutation into a cell line containing the
wild-type BCR-ABL sequence, in 10% increments. The wild-type sequence contains
an adenine at the nucleotide indicated by an arrow. The mutant sequence contains a
thymine at this position. The mutant was first reliably detected at a level of 20% and
became the predominant nucleotide at 70%. The N indicates that approximately equal
amounts of mutant and wild type were detected at a concentration of 50% and 60%.
PCR for these samples was repeated, the correct product was amplified. When
products are present after amplification of a smaller than expected size, the PCR
reaction should be repeated.
4. We have not found it necessary to calculate the concentration of the purified PCR
product before addition to the sequencing reaction. Visual analysis of the intensity
of the product on an agarose gel is adequate to determine the volume to add
to the reaction. The Big Dye sequencing chemistry is suitable for a broad range
of concentrations and 2 µL of product produces good quality sequencing for most