104 Gel & Capillary | RUO Assays Invivoscribe 2019 | 105 Gel & Capillary RUO Assays T-Cell Receptor Gamma Gene Rearrangement Assays Assay Use T-Cell Receptor Gamma Gene Rearrangement Assays are useful for studies involving: •  Identification of 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 Sample genomic DNA is amplified using two master mixes that independently target conserved regions within the variable (Vƴ) and joining (Jƴ) regions that flank the unique, hypervariable, antigen- binding, complementarity determining region 3 (CDR3). This assay targets Vƴ 1-9 and Jƴ gene segments. Positive and negative DNA controls, as well as an internal Amplification Control Master Mix, are included. The limit of detection of this assay is approximately one clonal T cell in a background of a hundred normal cells. PCR products can be analyzed by capillary electrophoresis or standard gel electrophoresis with ethidium bromide staining. Background The T-cell receptor gamma (TRG, formerly known as TCRG) chain locus spans 160 kilobases on chromosome 7 (7p14). The locus consists of 14 variable (Vƴ) gene segments in 6 subgroups, and 5 joining (Jƴ) gene segments interspersed between 2 constant (Cƴ) gene segments. However, the repertoire of functional TRG molecules is limited to 4-6 functional Vƴ gene segments that belong to 2 subgroups2 . Rearrangement of the Vƴ and Jƴ gene segments of the TRG locus results in Vƴ-Jƴ products of unique length and sequence. Clonal TRG rearrangements can be most rapidly identified by analyzing the size distribution of DNA products amplified from conserved sequences that flank this Vƴ-Jƴ region1 . DNA isolated from a normal heterogeneous population of polyclonal T-cells produces a Gaussian distribution (bell-shaped size curve) of amplified products. DNA amplified from a clonal T-cell population generates one or two product(s) of unique size that reflects proliferation of a single rearranged clone1,2 . 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.  JE Miller, SS Wilson, DL Jaye, and M Kronenberg. J. Mol. Diag. 4: 101-117 (1999). 2. K Beldjord et al., Leukemia 17: 2289-2292 (2003). Figure Legend: Simplified figure representing the organization of a rearranged T-cell receptor gamma chain gene on chromosome 7. Colored arrows represent conserved regions within the variable region gene segments targeted by primers. Primers are represented by arrows with the size range of valid products generated with each of the master mixes indicated below the figure. Colors correspond to the peak colors assigned to products when differential fluorescence detection methods are used. Reagents Ordering Information Catalog # Products Quantity 1-207-0010 T-Cell Receptor Gene Rearrangement Assay - Gel Detection 30 reactions 1-207-0030 T-Cell Receptor Gene Rearrangement Assay MegaKit - Gel Detection 300 reactions 1-207-0051 T-Cell Receptor Gene Rearrangement Assay - ABI Fluorescence Detection 30 reactions 1-207-0071 T-Cell Receptor Gene Rearrangement Assay MegaKit - ABI Fluorescence Detection 300 reactions Gel Electrophoresis Detection The data shown was generated using the master mix indicated. Amplified products were 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. 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-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 T-Cell Receptor Gamma Mix 1 Vƴ1-8,9 + Jƴ1/2 1 x 1500 μL tube 10 x 1500 μL tubes T-Cell Receptor Gamma Mix 2 Alt Vƴ + Jƴ1/2 1 x 1500 μL tube 10 x 1500 μL tubes Amplification Control HLA-DQa 1 x 1500 μL tube 10 x 1500 μL tubes