90 Gel & Capillary | RUO Assays Invivoscribe 2019 | 91 Gel & Capillary RUO Assays IGH Gene Rearrangement Assays Assay Use IGH Gene Rearrangement Assays are useful for studies involving: •  Identification of clonal B-cell populations highly suggestive of B-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 Genomic DNA is amplified using three PCR master mixes that target the three conserved framework regions (FR1, FR2, and FR3) of the IGH gene and the joining (JH) region. These regions flank the unique, hypervariable, antigen-binding, complementarity determining region 3 (CDR3). All positive and negative DNA controls, as well as an Amplification Control master mix, are included. The limit of detection of this assay is one clonal B 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. Clonality is indicated if one or more of the three framework master mixes generates clonal products. Background Genes encoding immunoglobulin heavy chain (IGH) molecules are assembled from multiple polymorphic gene segments that undergo rearrangement and selection during B-cell development2 . Rearrangement of these variable (VH), diversity (DH), and joining (JH) genetic segments result in VDJ products of unique length and sequence1,2 . Clonal IGH rearrangements can be rapidly identified through analyses of the size distributions of DNA products amplified from conserved sequences that flank this region1 . For example, DNA isolated from a normal polyclonal population of B cells produces a Gaussian distribution (bell-shaped size curve) of amplified products; whereas, DNA amplified from a clonal B-cell population generates one or two product(s) of unique size that reflect proliferation of a single rearranged clone1 . In comparison, southern blot analysis requires 1-2 weeks, is significantly less sensitive, and requires approximately one hundred times more DNA than PCR-based assays, which can be completed in 4-5 hours1 . In addition, tests of samples previously designated Quantity Not Sufficient (QNS), such as formalin- fixed, paraffin embedded (FFPE) tissue sections, routinely produce a valid result with PCR methods. 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 tissue or slides. Reference 1.  JE Miller, SS Wilson, DL Jaye, and M Kronenberg. J. Mol. Diag. 4: 101-117 (1999). 2. S Tonegawa. Nature 302: 575-581 (1983). Figure Legend: Genomic organization of a rearranged immunoglobulin heavy chain gene on chromosome 14. The blue and green arrows represent primers targeting the conserved framework regions within the variable region gene. The relative location, size range of valid products, and colors correspond to the products generated from each of these regions when differential fluorescence detection methods are used. Reagents Ordering Information Catalog # Products Quantity 1-101-0010 IGH Gene Rearrangement Assay - Gel Detection 30 reactions 1-101-0030 IGH Gene Rearrangement Assay MegaKit - Gel Detection 300 reactions 1-101-0051 IGH Gene Rearrangement Assay - ABI Fluorescence Detection 30 reactions 1-101-0071 IGH 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). IVS-0000 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-0030 Clonal Control DNA 200 μg/mL 1 x 100 μL tube 5 x 100 μL tubes IVS-0029 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 IGH Framework 1 Framework 1 + JH 1 x 1500 μL tube 10 x 1500 μL tubes IGH Framework 2 Framework 2 + JH 1 x 1500 μL tube 10 x 1500 μL tubes IGH Framework 3 Framework 3 + JH 1 x 1500 μL tube 10 x 1500 μL tubes Amplification Control HLA-DQa 1 x 1500 μL tube 10 x 1500 μL tubes