120 Gel & Capillary | RUO Assays Invivoscribe 2019 | 121 Gel & Capillary RUO Assays IGH Somatic Hypermutation Assays v2.0 Assay Use IGH Somatic Hypermutation Assays are useful for studies involving: •  Identifying clonal rearrangements of the immunoglobulin heavy chain (IGH) gene •  Assessing the extent of somatic hypermutation (SHM) in the variable (VH) gene sequence (IGHV) in patients with chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL) • Evaluating new research and methods in malignancy studies Summary and Explanation of the Test These assays amplify either genomic DNA or complementary DNA (cDNA) that lies between the upstream leader (VHL) or framework 1 (FR1) regions and the downstream joining (JH) region of the IGH gene. The assays employ two different master mixes: Hypermutation Mix 1 and Hypermutation Mix 2. The Hypermutation Mix 1 targets sequences between the leader (VHL) and joining (JH) regions. Therefore the amplicon product(s) span the entire variable (VH) region, which contains all framework (FR) and complementarity-determining regions (CDR). The Hypermutation Mix 2 targets sequences between the framework 1 (FR1) and joining (JH) regions. The resulting amplicons include a portion of the FR1 region to the downstream JH region. The primers that target the VHL and FR1 regions have been redesigned to include a universal sequencing tag at the 5-end. This new design allows for bi-directional sequencing of clonal PCR products with just one sequencing-tag specific forward primer and one JH reverse primer, thus ensuring a more reliable and complete coverage of clonal products. Current ERIC (European Research Initiative on CLL) guidelines recommend bi-directional sequencing when determining the IGH SHM status. Positive and negative DNA, positive RNA, as well as an amplification control are included in the assay. Clonality is indicated if any one of the master mixes generates clonal products. Background Immunoglobulin variable heavy chain gene hypermutation status provides important prognostic information for patients with chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL). The presence of IGH somatic hypermutation (SHM) is defined as greater or equal to 2% difference from the germline variable (VH) gene sequence, whereas less than 2% difference is considered evidence of no somatic hypermutation. This has clinical relevance, as there is a clear distinction in the median survival of patients with and without somatic hypermutation. Hypermutation of the IGHV gene is strongly predictive of a good prognosis while lack of mutation predicts a poor prognosis. This assay aids in identification, sequencing, and analysis of somatic hypermutation status of clonal products. PCR products can be analyzed by gel electrophoresis detection or by differential fluorescence detection using capillary electrophoresis followed by gel electrophoresis detection. PCR products are sequenced bidirectionally either directly, after gel extraction, or after cloning into a bacterial vector. The resulting sequence is then compared to IGH germline sequences to determine mutational status. Specimen Requirements These assays test genomic DNA or cDNA from the following sources: 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. 5 μg of total RNA or mRNA; or, 5. 1 μg of cDNA; or, 6. Formalin-fixed, paraffin-embedded (FFPE) tissue or slides. Reference 1. P Ghia et al., Leukemia 21: 1-3 (2007). 2. P Ghia et al., Blood 105: 1678-1685 (2005). 3. F Davi et al., Leukemia 22: 212-214 (2008). Reagents Figure Legend: Simple representation of the organization of a rearranged immunoglobulin heavy chain gene on chromosome 14. Black arrows represent the relative positions of primers that target the conserved Leader (L) and Framework 1 (FR1) regions, and the downstream consensus JH gene segments. Controls Concentration Units in Assay Units in Assay MegaKit IVS-0013 Clonal Control DNA 200 μg/mL 1 x 100 μL tube 5 x 100 μL tubes IVS-0013 Clonal Control RNA 400 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 Hypermutation Mix 1 v2.0 Leader + JH 1 x 1500 μL tube 10 x 1500 μL tubes Hypermutation Mix 2 v2.0 Framework 1 + JH 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 Primers Target Units in Assay Units in Assay MegaKit Primer - Hypermutation Leader + Framework 1 1 x 10 μL tube at 100 μM 5 x 10 μL tube IGH JH Primer JH 1 x 10 μL tube at 100 μM 5 x 10 μL tube Ordering Information Catalog # Products Quantity 5-101-0030 IGH Somatic Hypermutation Assay v2.0 - Gel Detection 33 reactions 5-101-0040 IGH Somatic Hypermutation Assay v2.0 MegaKit - Gel Detection 330 reactions 5-101-0031 IGH Somatic Hypermutation Assay v2.0 - ABI Fluorescence Detection 33 reactions 5-101-0041 IGH Somatic Hypermutation Assay v2.0 MegaKit - ABI Fluorescence Detection 330 reactions Gel Electrophoresis Detection The data shown was generated using the master mix indicated. Amplified products were run on a 2% agarose/TBE gel. Lane 1 is data generated testing cDNA synthesized from the recommended 100% clonal control RNA; 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 the IVS-0000 Polyclonal Control DNA (Cat# 4-092- 0010). Lanes flanking the samples were loaded with a 100 base pair DNA size standard. Capillary Electrophoresis Detection (ABI) The data shown was generated on an ABI 3130xl instrument using the master mix indicated. Panel 1 displays data generated testing cDNA synthesized from the recommended 100% clonal control RNA; panel 2 displays data generated testing the recommended 100% clonal control DNA; panel 3 displays data generated testing the recommended 10% clonal control DNA; and panel 4 displays data generated testing IVS-0000 Polyclonal Control DNA (Cat# 4-092-0010).