Rapid and Accurate Detection of Monoclonal Immunoglobulin Heavy Chain Gene Rearrangement by DNA Melting Curve Analysis in the LightCycler System Dongsheng.

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Presentation transcript:

Rapid and Accurate Detection of Monoclonal Immunoglobulin Heavy Chain Gene Rearrangement by DNA Melting Curve Analysis in the LightCycler System Dongsheng Xu, Juan Du, Cynthia Schultz, Ayesha Ali, Howard Ratech The Journal of Molecular Diagnostics Volume 4, Issue 4, Pages 216-222 (November 2002) DOI: 10.1016/S1525-1578(10)60706-4 Copyright © 2002 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Optimization of PCR cycles. DNA from 100% Namalwa B-cell line (N, top curve), a mixture of 50% Namalwa and 50% tonsil (0.5 N + 0.5 T, middle curve), and 100% tonsil (T, bottom curve) was amplified for 20 (A), 30 (B), 40 (C), 50 (D), and 60 (E) cycles. Namalwa started to produce a sharp −dF/dT peak after 30 PCR cycles (B). The 100% Namalwa/100% tonsil −dF/dT peak height ratio reached 3.88 at 40 cycles (C and Table 1). Additional cycles did not significantly improve this ratio. Therefore, we set the criteria for diagnosing a monoclonal B-cell DNA sample as having a −dF/dT peak height at least twice as high, and less than one-half as wide, as a polyclonal tonsil DNA sample after 40 cycles. x axis = temperature (°C); y axis = −dF/dT, where F = fluorescence and T = temperature. The Journal of Molecular Diagnostics 2002 4, 216-222DOI: (10.1016/S1525-1578(10)60706-4) Copyright © 2002 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 Minimal detection of B-cell clonality. Namalwa B-cell line DNA (50%, 25%, 12.5%, 6.25%, 3.13%, and 1.56%) were serially diluted into tonsil DNA. After 40 cycles of amplification in the LightCycler System, a distinct peak with the expected Tm of Namalwa was still detectable at the 12.5% level by DNA melting curve analysis. Axes definitions are the same as in Figure 1. The Journal of Molecular Diagnostics 2002 4, 216-222DOI: (10.1016/S1525-1578(10)60706-4) Copyright © 2002 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 Detecting B-cell clonality in representative clinical DNA samples using melting curve analysis. Sharp peaks above dashed line (−dF/dT = 0.35) indicate clonally rearranged IgH: Namalwa B-cell line and four-patient DNA samples12519 from three patients (see Table 2). DNA samples 1 and 2, obtained from a single individual, share the same Tm value indicating clonal identity. Broad peaks below dashed line (−dF/dT = 0.35) indicate polyclonal IgH rearrangements: tonsil and five unnumbered patient DNA samples. Axes definitions are the same as in Figure 1. The Journal of Molecular Diagnostics 2002 4, 216-222DOI: (10.1016/S1525-1578(10)60706-4) Copyright © 2002 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions