Consensus JH Gene Probes with Conjugated 3′-Minor Groove Binder for Monitoring Minimal Residual Disease in Acute Lymphoblastic Leukemia Michihiro Uchiyama,

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Consensus JH Gene Probes with Conjugated 3′-Minor Groove Binder for Monitoring Minimal Residual Disease in Acute Lymphoblastic Leukemia Michihiro Uchiyama, Chihaya Maesawa, Akiko Yashima-Abo, Mitsu Tarusawa, Mikiya Endo, Waka Sugawara, Shoichi Chida, Shima Onodera, Yasuhiko Tsukushi, Yoji Ishida, Shigeru Tsuchiya, Tomoyuki Masuda The Journal of Molecular Diagnostics Volume 7, Issue 1, Pages 121-126 (February 2005) DOI: 10.1016/S1525-1578(10)60017-7 Copyright © 2005 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Schematic diagram of the probe/primer design for the JH ASO RQ-PCR strategy. In this strategy, the sequencing analysis is performed using FR1c/LJH/VLJH and/or FR2a/LJH/VLJH primers to determine the V-D-J sequences. The number of the JH gene segment is used to choose an appropriate primer out of six intronic primers. A TaqMan MGB probe was designed for the 3′-end of the JH gene segments in combination with an allele-specific oligonucleotide (ASO) primer complementary to the junctional region and a primer complementary to the intronic sequence of the JH gene segments. The Journal of Molecular Diagnostics 2005 7, 121-126DOI: (10.1016/S1525-1578(10)60017-7) Copyright © 2005 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 Alignment of JH family sequences registered in the V BASE database. The regions of the JH non-MGB probe designed by Verhagen et al9 and our JH MGB probe are boxed. Dashes indicate identity with the germline sequence of JH4a, and differences are indicated by capital letters. The Journal of Molecular Diagnostics 2005 7, 121-126DOI: (10.1016/S1525-1578(10)60017-7) Copyright © 2005 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 Amplification plots and standard curves obtained using the MGB (A and C) and non-MGB probes (B). A: Amplification plot and standard curve for the MJHU probe. Amplification plots were constructed using genomic DNA from an ALL cell line (BALL 1). The cells were diluted with normal mononuclear cells: from 101 to 106 tumor cells in a total of 106 cells. The CT value for each dilution is indicated by an arrow (a–f). The standard curve was constructed using BALL 1-specific plasmid DNA (black dots, a–f). B: Amplification plot and standard curve constructed using a conventional TaqMan probe (non-MGB, JHQ1/4/5, see reference 9). Amplification plots were constructed using genomic DNA from an ALL cell line (BALL 1). The cells were diluted with normal mononuclear cells: from 101 to 106 tumor cells in a total of 106 cells. The CT value for each dilution is indicated by an arrow (a–f). The standard curve was constructed using BALL 1 specific-plasmid DNA (black dots and line). Serial dilutions of tumor cells are represented by clear circles (a–f). C: Amplification plot and standard curve constructed using the MJH2 probe. Amplification plots were constructed using genomic DNA from bone marrow samples at diagnosis (Patient 16 in Table 1). DNA was serially diluted in polyclonal DNA to give a final concentration of 10−1 to 10−6. The CT value for each dilution is indicated by an arrow (a–f). The standard curve was constructed using patient-specific plasmid DNA (black dots and line). Serial dilutions of bone marrow DNA are represented by clear circles (a–f). The Journal of Molecular Diagnostics 2005 7, 121-126DOI: (10.1016/S1525-1578(10)60017-7) Copyright © 2005 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 4 Normalized copy number of target genes before and after induction therapy using immunoglobulin heavy chain (IgH)-allele-specific oligonucleotide real-time quantitative polymerase chain reaction (ASO RQ-PCR) assay in two cases. RQ-PCR analysis with the MJHU probe (continuous line) is compared with the conventional assay using non-MGB probes (dotted line). The arrow indicates re-induction therapy for bone marrow transplantation. The Journal of Molecular Diagnostics 2005 7, 121-126DOI: (10.1016/S1525-1578(10)60017-7) Copyright © 2005 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions