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Rapid Detection of Clonal T-Cell Receptor-β Gene Rearrangements in T-Cell Lymphomas Using the LightCycler-Polymerase Chain Reaction with DNA Melting Curve.

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Presentation on theme: "Rapid Detection of Clonal T-Cell Receptor-β Gene Rearrangements in T-Cell Lymphomas Using the LightCycler-Polymerase Chain Reaction with DNA Melting Curve."— Presentation transcript:

1 Rapid Detection of Clonal T-Cell Receptor-β Gene Rearrangements in T-Cell Lymphomas Using the LightCycler-Polymerase Chain Reaction with DNA Melting Curve Analysis  Xiao Yan Yang, Dongsheng Xu, Juan Du, Hideko Kamino, Jennifer Rakeman, Howard Ratech  The Journal of Molecular Diagnostics  Volume 7, Issue 1, Pages (February 2005) DOI: /S (10) Copyright © 2005 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

2 Figure 1 Optimization of PCR cycles. DNA samples from 100% Jurkat T-cell line (J, top curve), a mixture of 50% Jurkat and 50% tonsil (0.5 J T, middle curve), 100% tonsil (T, bottom curve), and a negative control including all reagents without DNA (H2O) were amplified for 20 (A), 30 (B), 40 (C), 50 (D), and 60 (E) cycles. B: Jurkat started to produce a sharp −dF/dT peak after 30 PCR cycles. D: The −dF/dT peak height ratio of 100% Jurkat/100% tonsil reached 4.1 at 50 cycles (Table 1). Additional cycles did not significantly improve this ratio. Therefore, the diagnosis of a clonal T-cell gene rearrangement sample was based on DNA melting after 50 PCR cycles. x axis = temperature, ° C; y axis = −dF/dT, where F = fluorescence and T = temperature. The Journal of Molecular Diagnostics 2005 7, 81-88DOI: ( /S (10) ) Copyright © 2005 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

3 Figure 2 Minimal detection of percent clonal T cell. Jurkat T-cell line DNA (100%, 50%, 25%, 12.5%, 6.25%, and 3.125%) was serially diluted into tonsil DNA. After 50 cycles of amplification in the LightCycler system, we could still detect a distinct peak with the expected Tm of Jurkat by DNA melting curve analysis at the 12.5% level with primer set A (A) and at 6.25% level with primer set C (B). The Journal of Molecular Diagnostics 2005 7, 81-88DOI: ( /S (10) ) Copyright © 2005 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

4 Figure 3 Detecting T-cell clonality in representative clinical DNA samples by melting curve analysis using primer set A (A) and primer set C (B). Sharp peaks above the dashed line (−dF/dT = 0.85 for A and 0.28 for B) indicate clonally rearranged TCR-β: Jurkat T-cell line (J), CEM T-cell line (C), and three patient DNA samples (A and Table 4) and four patient DNA samples (B and Table 4). Broad peaks under the dashed line indicate polyclonal TCR-β gene rearrangements in tonsil and two representative unnumbered patient DNA samples. Axes definitions are the same as in Figure 1. The Journal of Molecular Diagnostics 2005 7, 81-88DOI: ( /S (10) ) Copyright © 2005 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

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