Ming-Tseh Lin, Li-Hui Tseng, Roy G. Rich, Michael J

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Presentation transcript:

Δ-PCR, A Simple Method to Detect Translocations and Insertion/Deletion Mutations Ming-Tseh Lin, Li-Hui Tseng, Roy G. Rich, Michael J. Hafez, Shuko Harada, Kathleen M. Murphy, James R. Eshleman, Christopher D. Gocke The Journal of Molecular Diagnostics Volume 13, Issue 1, Pages 85-92 (January 2011) DOI: 10.1016/j.jmoldx.2010.11.004 Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Primer design for detection of BCL2-IGH translocation (A) and internal tandem deletion mutation of the FLT3 gene (B). Sizes are not to scale. Arrows indicate primers and lines indicates location of the probe for real-time PCR. The Δ indicates the defined size difference of the paired amplicons for each breakpoint and an asterisk indicates the segment of ITD mutation. The Journal of Molecular Diagnostics 2011 13, 85-92DOI: (10.1016/j.jmoldx.2010.11.004) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Analytic sensitivity of Δ-PCR for BCL2-IGH translocation. Genomic DNA (250 pg, or 40–50 cell equivalents) from cell lines SU-DHL-6 (A), SU-DHL-4 (B), and SU-DHL-16 (C) (positive control for MBR1, MBR2, and mcr2 breakpoints, respectively) was subjected to multiplex Δ-PCR and analyzed by electrophoresis (ABI 3100 system, Applied Biosystems, Foster City, CA) with 45-second electrokinetic injection. Blue peaks are Δ-PCR products (Δ numbers indicate amplicon size difference in base pairs) and red peaks are size markers. The presence of two peaks with size difference of Δ18 ± 1 bp, Δ24 ± 1 bp, and Δ33 ± 1 bp indicates BCL2-IGH translocation with breakpoints at MBR1, MBR2, and mcr2, respectively. The Journal of Molecular Diagnostics 2011 13, 85-92DOI: (10.1016/j.jmoldx.2010.11.004) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Representative cases with MBR1, mcr1, and mcr2 breakpoints detected by Δ-PCR. DNA (20 ng) extracted from cases 36 (A), 19 (B), and 30 (C) and from a case with lymphoid hyperplasia (D) was subjected to multiplex Δ-PCR and analyzed by capillary electrophoresis with 45-second electrokinetic injection. Cases 36, 19, and 30 were positive for MBR1 (Δ18 ± 1 bp), mcr1 (Δ39 ± 1 bp), and mcr2 (Δ33 ± 1 bp), respectively, based on the size difference of the two amplicon peaks. The Journal of Molecular Diagnostics 2011 13, 85-92DOI: (10.1016/j.jmoldx.2010.11.004) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Detection of multiple minor leukemic clones with internal tandem duplication mutation by Δ-PCR. The two wild-type peaks were 328 bases (blue) and 353 bases (green). Four mutant clones (M1, M2, M3, and M4) were detected. Each clone has one blue peak and one green peak, with a Δ value of 25 bases. The peak height ratio of M4/M2 was 6.4%, ratio of M2/wild-type was 6.2%, and the ratio of M4/wild-type was 0.4% (6.4% × 6.2%). *The red striped line within the 353-base wild-type peak indicates off-scale height. The PCR product was therefore diluted fivefold and reinjected into the ABI 3100 genetic analyzer (data not shown) to calculate the ratio of M2/wild-type. The Journal of Molecular Diagnostics 2011 13, 85-92DOI: (10.1016/j.jmoldx.2010.11.004) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions