BCR/ABL, Tyrosine Kinase Inhibitor Resistance, Kinase Domain Mutation Screen
Method Description Describes how the test is performed and provides a method-specific reference
Total RNA is extracted from the sample using the miRNeasy extraction kit (Qiagen). cDNA is made using the High Capacity cDNA Kit (Applied Biosystems). A first-round PCR is performed using primers directed against BCR and ABL regions to generate a PCR product representing the translocated allele only (p210, p190, p185 or p230 transcript types). A second (nested) PCR is next performed to amplify the ABL kinase domain region using template from the first-round PCR product. Aliquots of the ABL PCR are subjected to multiplex allele-specific primer extension (ASPE) reactions using primers specific for the targeted mutation sites (M244V, G250E, Q252H [G->C, G->T], Y253F, Y253H, E255K, T315I, F317L [C->G, C->A, T->C], M351T, E255G, F359V, and H396R). The ASPE reactions which have biotin-dCTP incorporated are incubated with microsphere beads containing specific recognition tag sequences (FlexMap, Luminex) and streptavidin-phycoerythrin is added to label any bound ASPE sequences. The target mutations are recognized following flow cytometry by specific bead address and fluorescence intensity.(Unpublished Mayo method)
Day(s) and Time(s) Test Performed Outlines the days and times the test is performed. This field reflects the day and time the sample must be in the testing laboratory to begin the testing process and includes any specimen preparation and processing time required before the test is performed. Some tests are listed as continuously performed, which means assays are performed several times during the day.
Monday through Friday