100 Gel & Capillary | RUO Assays Invivoscribe 2019 | 101 Gel & Capillary RUO Assays TCRB Gene Clonality Assays Assay Use TCRB Gene Clonality Assays are useful for studies involving: •  Identification clonal T-cell populations highly suggestive of T-cell malignancies • Lineage determination of leukemias and lymphomas • Monitoring and evaluation of disease recurrence • Detection and assessment of residual disease • Evaluation of new research and methods in malignancy studies Summary and Explanation of the Test Three multiplex master mixes target conserved regions within the variable (Vβ), diversity (Dβ), and the joining (Jβ) regions that flank the unique hypervariable, antigen-binding, complementarity determining region 3 (CDR3) of the T-cell receptor beta locus. Tube A contains 23 Vβ primers, 6 Jβ1 primers, and 3 Jβ2 primers. Tube B contains 23 Vβ and 4 Jβ2 primers. Tube C contains 2 Dβ and 13 Jβ primers. Positive and negative DNA controls, as well as a Specimen Control Size Ladder Master Mix, are included. PCR products can be analyzed by capillary electrophoresis or heteroduplex analysis. Clonality is indicated, if any one of the master mixes generates clonal products. Background The human T-cell receptor beta (TRB, formerly known as TCRB) gene locus on chromosome 7 (7q34, formerly 7q35) includes 64-67 variable (Vβ) gene segments (belonging to 30 subgroups), 2 diversity (Dβ) gene segments, and 13 joining (Jβ) gene segments, spread over 685 kilobases. The diversity of this locus has complicated PCR-based testing and extended dependence on Southern blot analysis in many testing centers. However, this standardized multiplex PCR assay detects the vast majority of clonal TRB gene rearrangements using only three multiplex master mixes1 . This assay provides rapid TCR clonality assessment, reducing or completely eliminating the number of Southern blot tests performed in the laboratory. The detection rate of clonal TRB gene rearrangements using this assay is exceptionally high1 . The performance characteristics of this assay have been independently determined by the EuroClonality/BIOMED-2 Group. This consortium of 47 molecular diagnostic laboratories tested hundreds of clinical samples, taking into account their lymphoproliferative status as defined by the WHO classifications2 . Specimen Requirements 1.  5 mL of peripheral blood, bone marrow biopsy, or bone marrow aspirate anti-coagulated with heparin or EDTA; or, 2. Minimum 5 mm cube of tissue; or, 3. 2 µg of genomic DNA; or, 4. Formalin-fixed, paraffin-embedded (FFPE) tissue or slides. Reference 1. M Brüggemann et al., Leukemia 17: 2283-2289 (2003). 2. JJM van Dongen et al., Leukemia 17: 2257-2317 (2003). Reagents Ordering Information Catalog # Products Quantity 1-205-0010 TCRB Gene Clonality Assay - Gel Detection 33 reactions 1-205-0020 TCRB Gene Clonality Assay MegaKit - Gel Detection 330 reactions 1-205-0011 TCRB Gene Clonality Assay - ABI Fluorescence Detection 33 reactions 1-205-0021 TCRB Gene Clonality Assay MegaKit - ABI Fluorescence Detection 330 reactions Gel Electrophoresis Detection The data shown was generated using the master mix indicated. Amplified products were heteroduplexed and run on a 6% non-denaturing polyacrylamide/TBE gel. Lane 1 is data generated testing an alternative 100% clonal control DNA; lane 2 is data generated testing the recommended 100% clonal control DNA; lane 3 is data generated testing a 10% dilution of the recommended clonal control DNA; and, lane 4 is data generated testing IVS-0000 Polyclonal Control DNA (Cat# 4-092- 0010). The IVS-0000 control is used as a negative control for many of our tests. A standard 100 base pair DNA size ladder was run in the lanes flanking the test samples. Capillary Electrophoresis Detection (ABI) The data shown was generated using the master mix indicated. Amplified products were run on an ABI 3100 instrument. Panel 1 displays data generated testing an alternative 100% clonal control DNA; panel 2 displays data generated testing the recommended 100% clonal control DNA; panel 3 displays data generated testing a 10% dilution of the recommended clonal control DNA; and, panel 4 displays data generated testing the IVS-0000 Polyclonal Control DNA (Cat# 4-092-0010). Our IVS-0000 DNA is often tested to provide information regarding valid size ranges for each master mix. Figure Legend: Simplified diagram of a representative rearranged T-cell receptor beta gene showing the approximate placement of the upstream and downstream DNA primers. The numbers of primers and their specificity are listed for Master Mix Tubes A, B, and C. Controls Concentration Units in Assay Units in Assay MegaKit IVS-0009 Clonal Control DNA 200 μg/mL 1 x 100 μL tube 5 x 100 μL tubes IVS-0004 Clonal Control DNA 200 μg/mL 1 x 100 μL tube 5 x 100 μL tubes IVS-0000 Polyclonal Control DNA 200 μg/mL 1 x 100 μL tube 5 x 100 μL tubes Master Mixes Target Units in Assay Units in Assay MegaKit TCRB Tube A Multiple Vβ + Jβ1/2 1 x 1500 μL tube 10 x 1500 μL tubes TCRB Tube B Multiple Vβ + Jβ2 1 x 1500 μL tube 10 x 1500 μL tubes TCRB Tube C Multiple Dβ + Jβ1/2 1 x 1500 μL tube 10 x 1500 μL tubes Specimen Control Size Ladder Multiple Genes 1 x 1500 μL tube 10 x 1500 μL tubes This assay is based on the EuroClonality/BIOMED-2 Concerted Action BMH4-CT98-3936.