Detection of FLT3 Internal Tandem Duplication and D835 Mutations by a Multiplex Polymerase Chain Reaction and Capillary Electrophoresis Assay Kathleen.

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Detection of FLT3 Internal Tandem Duplication and D835 Mutations by a Multiplex Polymerase Chain Reaction and Capillary Electrophoresis Assay Kathleen M. Murphy, Mark Levis, Michael J. Hafez, Tanya Geiger, Lisa C. Cooper, B.Douglas Smith, Donald Small, Karin D. Berg The Journal of Molecular Diagnostics Volume 5, Issue 2, Pages 96-102 (May 2003) DOI: 10.1016/S1525-1578(10)60458-8 Copyright © 2003 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Diagram of the assay design. The FLT3 gene consists of five extracellular immunoglobulin-like domains, a transmembrane domain (TM), a JM domain, and an interrupted kinase domain (TK1 and TK2). PCR primers flanking the JM domain [forward primer labeled with FAM (blue), reverse primer labeled with NED (yellow)] and primers specific for the TK2 domain [forward labeled with TET (green), reverse unlabeled] are multiplexed into a single PCR reaction. After amplification, the PCR products are digested with EcoRV. The dotted lines in the TK2 PCR product represent the EcoRV cut sites, with the recognition sequence (GATATC). The JM portion of the PCR yields a wild-type PCR product of 330 bases labeled with both FAM and NED. FLT3 ITD mutations result in PCR products that are longer than wild type (>330 bp), also labeled with both FAM and NED. After digestion, the D835 portion of the assay yields wild-type products sizing at 80 bases that are TET labeled. D835 mutant TET-labeled products size at 129 bases, and undigested TET-labeled products size at 150 bases. The Journal of Molecular Diagnostics 2003 5, 96-102DOI: (10.1016/S1525-1578(10)60458-8) Copyright © 2003 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 Examples of results of the FLT3 assay. A–D: CE pherograms; x axis represents size of the PCR products in bases, y axis represents relative fluorescence intensity. Red peaks represent internal size standard. Green (TET) PCR product peaks result from the D835 portion of the assay. Blue (FAM) and black (NED) peaks result form the ITD portion of the assay. A: Example of a FLT3 D835 wild-type/ITD wild-type result. B: Example of a FLT3 D835 wild-type/ITD mutant result. C: Example of a D835 mutant/ITD wild-type result. D: Example of an incomplete digest for the D835 assay and a wild-type ITD. The Journal of Molecular Diagnostics 2003 5, 96-102DOI: (10.1016/S1525-1578(10)60458-8) Copyright © 2003 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 Example of enrichment of a sample containing a small number of FLT3 ITD mutant allele PCR products because of a low blast count in the sample tested. A: CE pherogram; x axis represents size of the PCR product in bases, y axis represents relative fluorescence intensity. Red peaks represent internal size standard. Blue/black peaks are the FAM/NED-labeled products of the ITD portion of the multiplexed assay. The large wild-type peak at 330 bases and small ITD mutant peak at 354 bases are indicated by black and red arrows, respectively. B: PAGE of the same PCR product as in A. Lane 1 is a 50-bp marker. Lane 2 is the same sample analyzed by CE in A. The black arrow indicates the 330-bp wild-type product, and the red arrow indicates the 354-bp ITD mutant PCR product. C: CE electropherogram after band-stab of the ITD mutant band and reamplification. Note that after band-stab and reamplification, the FLT3 ITD mutant PCR products are dominant compared to wild type. The Journal of Molecular Diagnostics 2003 5, 96-102DOI: (10.1016/S1525-1578(10)60458-8) Copyright © 2003 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 4 Example of improved resolution of CE compared to PAGE. A: CE pherogram of a sample positive for two different ITD mutations; x axis represents size of the PCR product in bases, y axis represents relative fluorescence intensity. Red peaks represent the internal size standard. The wild-type peak at 330 bases (black arrow) and two ITD mutant peaks at 348 and 351 bases (red arrows) are indicated. Below, the electropherogram is magnified to highlight the two different ITD mutations. B: PAGE of the same PCR product as in A. Lane 1 is a 50-bp marker, lane 2 is a no template control, lane 3 is a FLT3 wild-type control, lane 4 is a FLT3 ITD control, and lane 5 is the same sample as run by CE in A. The black arrow indicates the small amount of wild-type PCR product. The red arrow indicates the two ITD bands at 348 and 351 identified by CE. The arrowheads identify bands that are not detected by CE. These bands appear to be the result of heteroduplex formation between the two ITD products and between wild-type and ITD PCR products (see text). The Journal of Molecular Diagnostics 2003 5, 96-102DOI: (10.1016/S1525-1578(10)60458-8) Copyright © 2003 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions