b"Gel & Capillary | RUO AssaysGel and Capillary Gel and CapillaryIGH Somatic Hypermutation Assays v2.0 Research Use Only (RUO) Assay IGH Somatic Hypermutation Assays v2.0 Research Use Only (RUO) AssayIGH Somatic Hypermutation Assays v2.0Assay Use BackgroundReagentsThe Research Use Only IGH Somatic Hypermutation Assay v2.0 is usedRearrangements of the antigen receptor genes occur during ontogenyControls Concentration Units in Assay Units in Assay MegaKitto identify clonal rearrangements of the immunoglobulin heavy (IGH)in B and T lymphocytes.These gene rearrangements are unique chain gene and determine the somatic mutation status of the variablein length and sequence for each cell. Therefore, polymerase chainIVS-0013 Clonal Control DNA 200 g/mL 1 x 100 L tube 5 x 100 L tubes(V) gene sequence andis useful for the study of: reaction (PCR) assays can be used to identify lymphocyte populationsIVS-0013 Clonal Control RNA 400 g/mL 1 x 100 L tube 5 x 100 L tubesderived from a single cell by detecting the unique V-J gene Identifying clonal rearrangements of the IGH chain gene rearrangements present within these antigen receptor loci. 1This PCR- IVS-0000 Polyclonal Control DNA 200 g/mL 1 x 100 L tube 5 x 100 L tubesAssessing the extent of somatic hypermutation in the variablebased assay employs multiple consensus DNA primers that targetMaster Mixes Target Units in Assay Units in Assay MegaKitregion of the immunoglobulin heavy chain gene conserved genetic regions within the immunoglobulin heavy chain Evaluating new research and methods in malignancy studies (IGH) gene.This test is used to detect and sequence the majorityHypermutation Mix 1 v2.0 Leader + J H 1 x 1500 L tube 10 x 1500 L tubesof clonal IGH rearrangements from either genomic DNA (gDNA) orHypermutation Mix 2 v2.0 Framework 1 + J H 1 x 1500 L tube 10 x 1500 L tubescomplementary DNA (cDNA). Clonal products can be detected using Summary and Explanation of the Testa variety of methods, including gel and capillary electrophoresis. Specimen Control Size Ladder Multiple Genes 1 x 1500 L tube 10 x 1500 L tubesThese assays amplify either genomic DNA or complementary DNAThe presence of IGH somatic hypermutation (SHM) is defined asPrimers Target Units in Assay Units in Assay MegaKit(cDNA) that lies between the upstream leader (V H L) or framework 1greater or equal to 2% difference from the germline variable (V) genePrimer - Hypermutation Leader + Framework 1 1 x 10 L tube at 100 M 5 x 10 L tubesequence, whereas less than 2% difference is considered evidence of (FR1) regions and the downstream joining (J H ) region of the IGH gene.no somatic hypermutation. 3 HH 5 x 10 The assays employ two different master mixes: Hypermutation Mix 1IGH J Primer J 1 x 10 L tube at 100 M L tubeand Hypermutation Mix 2. The Hypermutation Mix 1 targets sequences between the leader (V H L) and joining (J H ) regions. Therefore theSpecimen Requirementsamplicon product(s) span the entire variable (V H ) region, which contains all framework (FR) and complementarity-determiningThis assay tests extracted and purified genomic DNA (gDNA).regions (CDR). The Hypermutation Mix 2 targets sequences betweenOrdering Informationthe framework 1 (FR1) and joining (J H ) regions. The resulting amplicons include a portion of the FR1 region to the downstream J Hregion. TheReferences Catalog # Products Quantityprimers that target the V H L and FR1 regions have been redesigned to1.P Ghia et al., Leukemia 21: 1-3 (2007).include a universal sequencing tag at the 5'end. This design allows2. P Ghia et al., Blood 105: 1678-1685 (2005). 5-101-0030 IGH Somatic Hypermutation Assay v2.0 - Gel Detection 33 reactionsfor bi-directional sequencing of clonal PCR products with just one3. F Davi et al., Leukemia 22: 212-214 (2008). 5-101-0040 IGH Somatic Hypermutation Assay v2.0 MegaKit - Gel Detection 330 reactionssequencing-tag specific forward primer and one J Hreverse primer, thus ensuring a more reliable and complete coverage of clonal5-101-0031 IGH Somatic Hypermutation Assay v2.0 - ABI Fluorescence Detection 33 reactionsproducts. Positive and negative DNA, positive RNA, as well as an5-101-0041 IGH Somatic Hypermutation Assay v2.0 MegaKit - ABI Fluorescence Detection 330 reactionsamplification control are included in the assay. Clonality is indicated if any one of the master mixes generates clonal products.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 J Hgene segments.124Invivoscribe 2021|125"